Compositions and methods related to engineered erythoid cells comprising 4-1BBL

ABSTRACT

The invention includes compositions and methods related to multimodal therapies, e.g., for treating a cancer. A multimodal therapy described herein provides and/or administers a plurality of agents that function in a coordinated manner to provide a therapeutic benefit to a subject in need thereof, e.g., a subject having a cancer.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2017/013035, filed Jan. 11, 2017, which claims priority to U.S.Ser. No. 62/277,130 filed Jan. 11 2016, U.S. Ser. No. 62/359,448 filedJul. 7, 2016, U.S. Ser. No. 62/370,915 filed Aug. 4, 2016, and U.S. Ser.No. 62/420,973 filed Nov. 11, 2016, the contents of which areincorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 11, 2018, isnamed R2081-702020FT_SL.txt and is 26,907 bytes in size.

BACKGROUND

Red blood cells have been considered for use as drug delivery systems,e.g., to degrade toxic metabolites or inactivate xenobiotics, and inother biomedical applications.

SUMMARY OF THE INVENTION

The invention includes compositions and methods related to multimodaltherapies. The therapies are useful, e.g., for treating cancer. Amultimodal therapy described herein provides and/or administers aplurality of agents that function in a coordinated manner to provide atherapeutic benefit to a subject in need thereof, e.g., a subject havinga cancer. In general, a multimodal therapy described herein includes theadministration to a subject of a preparation of engineered red bloodcells, e.g., enucleated red blood cells, comprising (e.g., expressing orcontaining) a plurality of agents (e.g., polypeptides) that function ina coordinated manner (e.g., agent-additive, agent-synergistic,multiplicative, independent function, localization-based,proximity-dependent, scaffold-based, multimer-based, or compensatory).

In some aspects, the present disclosure provides an enucleated red bloodcell, e.g., a reticulocyte, comprising a plurality of agents, e.g., aplurality of polypeptides (e.g., exogenous polypeptides), e.g., a firstexogenous polypeptide, a second exogenous polypeptide, and a thirdexogenous polypeptide.

In some aspects, the present disclosure provides an enucleated red bloodcell, e.g., a reticulocyte, comprising a plurality of exogenouspolypeptides, wherein a first and a second exogenous polypeptide of theplurality have agent-additive, agent-synergistic, multiplicative,independent function, localization-based, proximity-dependent,scaffold-based, multimer-based, or compensatory activity.

In some aspects, the present disclosure provides an enucleated red bloodcell, e.g., a reticulocyte, comprising a first exogenous polypeptide anda second exogenous polypeptide, wherein:

-   -   a) the first and second exogenous polypeptides act on the same        target, wherein optionally the target is a cell surface receptor        and/or an endogenous human protein;    -   b) the first exogenous polypeptide binds to a first endogenous        human protein and the second exogenous polypeptide binds to a        second endogenous human target protein, e.g., with a Kd of less        than 500, 200, 100, 50, 20, 10, 5, 2, or 1 nM;    -   c) the first exogenous polypeptide acts on (e.g., binds) a first        target, and the second exogenous polypeptide act on (e.g.,        binds) a second target, wherein the first and second targets are        members of the same biological pathway, wherein optionally the        targets are cell surface receptors, endogenous human proteins,        or both;    -   d) the first exogenous polypeptide comprises a first        pro-apoptotic polypeptide and the second exogenous polypeptide        comprises a second pro-apoptotic polypeptide, e.g., a TRAIL        receptor ligand, e.g., a TRAIL polypeptide;    -   e) the first and second exogenous polypeptides are in close        proximity to each other, e.g., are less than 10, 7, 5, 4, 3, 2,        1, 0.5, 0.2, or 0.1 nm apart for a duration of at least 1, 2, 5,        10, 30, or 60 seconds; 1, 2, 5, 10, 30, or 60 minutes, or 1, 2,        3, 6, 12, or 14 hours;    -   f) the first and second exogenous polypeptides have a Kd of less        than 500, 200, 100, 50, 20, 10, 5, 2, or 1 nM for each other;    -   g) the first exogenous polypeptide comprises an        antigen-presenting polypeptide, e.g., an MHC molecule, e.g., an        MHC class II molecule, and the second exogenous polypeptide        comprises an antigen, e.g., a cancer antigen;    -   h) the first and second exogenous polypeptides act on different        targets, wherein optionally at least one of the targets is a        cell surface receptor and/or an endogenous human protein, e.g.,        the first exogenous polypeptide binds a first cell type, e.g., a        cancer cell, and the second exogenous polypeptide binds a second        cell type, e.g., an immune effector cell, e.g., a T cell;    -   i) the first exogenous polypeptide and the second exogenous        polypeptide have an abundance ratio of about 1:1, from about 2:1        to 1:2, from about 5:1 to 1:5, from about 10:1 to 1:10, from        about 20:1 to 1:20, from about 50:1 to 1:50, from about 100:1 to        1:100 by weight or by copy number;    -   j) the first exogenous polypeptide and the second exogenous        polypeptide have a Kd for a first target and a second target,        respectively, with a ratio of about 1:1, from about 2:1 to 1:2,        from about 5:1 to 1:5, from about 10:1 to 1:10, from about 20:1        to 1:20, from about 50:1 to 1:50, from about 100:1 to 1:100;    -   k) the first exogenous polypeptide has a first activity (e.g.,        binding) towards a first target, and the second exogenous        polypeptide has a second activity (e.g., binding) towards the        first target, e.g., the first and second exogenous polypeptides        bind a single target;    -   l) the first exogenous polypeptide acts on (e.g., binds) a first        target and the second exogenous polypeptide acts on (e.g.,        binds) a second target, and the first and second targets are        part of the same pathway, wherein optionally the first exogenous        polypeptide acts on the first target and the second exogenous        polypeptide acts on the second target simultaneously;    -   m) the first exogenous polypeptide acts on (e.g., binds) a first        target and the second exogenous polypeptide acts on (e.g.,        binds) a second target, and the first and second targets are        part of different pathways, wherein optionally the first and        second pathways both act to promote a given cellular response;    -   n) the first exogenous polypeptide localizes the enucleated red        blood cell to a desired site, e.g., a human cell, and the second        exogenous polypeptide has a therapeutic activity, e.g., an        immunomodulation activity such as a T cell activation activity        or antigen presenting activity (e.g., for a cancer vaccine);    -   o) the first exogenous polypeptide binds a first target, e.g., a        first cell, e.g., a first cell type, e.g., a cancer cell, and        the second exogenous polypeptide binds a second target, e.g., a        second cell, e.g., a second cell type, e.g., an immune effector        cell, e.g., a T cell;    -   p) the first exogenous polypeptide and the second exogenous        polypeptide are non-human proteins;    -   q) the first exogenous polypeptide and the second exogenous        polypeptide are both enzymes, e.g., biosynthetic enzymes;    -   r) the first exogenous polypeptide promotes formation of an        intermediate molecule and the second exogenous polypeptide acts        on the intermediate molecule; or    -   s) the first exogenous polypeptide and the second exogenous        polypeptide act on successive steps of a pathway.

Any of the aspects herein, e.g., the aspects above, can be characterizedby one or more of the embodiments herein, e.g., the embodiments below.

In some embodiments, the exogenous polypeptides have synergisticactivity. In some embodiments, the exogenous polypeptides have additiveactivity.

In some embodiments, the exogenous polypeptides have proximity-dependentactivity. The proximity between the plurality of polypeptides, before,during, or after, interaction with a target moiety or moieties, mayconfer a property or result which is not seen in the absence of suchproximity in vivo or in vitro.

In some embodiments, the first exogenous polypeptide interacts with,e.g., binds, a first target moiety, e.g., a first target cellpolypeptide on a target cell (e.g., an immune effector cell, e.g., a Tcell), and the second exogenous polypeptide interacts with, e.g., binds,a second target moiety, e.g., a second target cell polypeptide on thetarget cell (e.g., wherein binding of the first and second target cellpolypeptide alters a biological property of the target cell). In anembodiment the first and second targets are subunits of a multimericcomplex on the target cell.

In some embodiments, the first exogenous polypeptide promotes fusion ofthe red blood cell with a target cell and the second exogenouspolypeptide is a polypeptide of any of Table 1, Table 2, or Table 3(e.g., a human polypeptide of any of Table 1, Table 2, Table 3, or Table4, e.g., a polypeptide having the amino acid sequence of the human wildtype polypeptide).

In some embodiments the first and second exogenous polypeptides interactwith one another, e.g., the first modifies, e.g., by cleavage orphosphorylation, the second, or the first and second form a dimeric ormultimeric protein.

In some embodiments, the enucleated red blood cell comprises 3, 4, 5, 6,7, 8, 9, or 10 different exogenous polypeptides. In an embodiment aplurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10), or all, of thedifferent exogenous polypeptides, have a preselected level of homologyto each other, e.g., at least 40, 50, 60, 70, 75, 80, 85, 90, 95, 96,97, 98, 99, or 99.5% sequence identity to each other. In an embodiment aplurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10), or all, of thedifferent exogenous polypeptides, have a preselected level of homologyto a reference sequence, e.g., at least 40, 50, 60, 70, 75, 80, 85, 90,95, 96, 97, 98, 99, 99.5%, or 100% sequence identity with a referencesequence (which reference sequence, includes an entire polypeptidesequence, or a portion thereof, e.g., a preselected domain), e.g., aplurality or all of the different exogenous polypeptides are antibodiesor antibody molecules. In some embodiments, the reference sequence is anantibody sequence or fragment thereof. In some embodiments, thereference sequence comprises a heavy chain constant region or portionthereof, light chain constant region or fragment thereof, heavy chainvariable region or portion thereof, light chain variable region orfragment thereof, or any combination of the foregoing.

In some embodiments, the enucleated red blood cell comprises at least 2but no more than 5, 6, 7, 8, 9, or 10 different exogenous polypeptides,e.g., exogenous polypeptides that are encoded by one or more exogenousnucleic acids that are not retained by the enucleated red blood cell.

In some embodiments, the exogenous polypeptides are encoded by one ormore exogenous nucleic acids that are not retained by the enucleated redblood cell.

In some embodiments, one or more (e.g., two or three) of the first,second, and optionally third exogenous polypeptides are transmembranepolypeptides or surface-anchored polypeptides.

In some embodiments, the first exogenous polypeptide interacts with,e.g., binds, a moiety on a target cell, and the second exogenouspolypeptide alters a property of the target cell, e.g., kills oractivates the target cell.

In some embodiments, the first exogenous polypeptide and the secondexogenous polypeptide have an abundance ratio of about 1:1, from about2:1 to 1:2, from about 5:1 to 1:5, from about 10:1 to 1:10, from about20:1 to 1:20, from about 50:1 to 1:50, or from about 100:1 to 1:100 byweight or by copy number. In some embodiments, both the first and secondpolypeptides have a stoichiometric mode of action, or both have acatalytic mode of action, and both are present at a similar abundance,e.g., about 1:1 or from about 2:1 to 1:2. In some embodiments, the firstexogenous polypeptide is more abundant than the second exogenouspolypeptide by at least about 10%, 20%, 30%, 50%, or a factor of 2, 3,4, 5, 10, 20, 50, or 100 (and optionally up to 10 or 100 fold) by weightor copy number. In some embodiments, the second exogenous polypeptide ismore abundant than the first exogenous polypeptide by at least about10%, 20%, 30%, 50%, or a factor of 2, 3, 4, 5, 10, 20, 50, or 100 (andoptionally up to 10 or 100 fold) by weight or copy number. In someembodiments, the first polypeptide has a stoichiometric mode of actionand the second polypeptide has a catalytic mode of action, and the firstpolypeptide is more abundant than the second polypeptide. In someembodiments, the second polypeptide has a stoichiometric mode of actionand the first polypeptide has a catalytic mode of action, and the secondpolypeptide is more abundant than the first polypeptide.

In some embodiments, the first exogenous polypeptide comprises atargeting moiety.

In some embodiments, the enucleated red blood cell has one or more ofthe following characteristics:

-   -   a) an osmotic fragility of less than 50% cell lysis at 0.3%,        0.35%, 0.4%, 0.45%, or 0.5% NaCl;    -   b) a cell volume of about 10-200 fL or a cell diameter of        between about 1 micron and about 20 microns, between about 2        microns and about 20 microns, between about 3 microns and about        20 microns, between about 4 microns and about 20 microns,        between about 5 microns and about 20 microns, between about 6        microns and about 20 microns, between about 5 microns and about        15 microns, or between about 10 microns and about 30 microns;    -   c) greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% fetal        hemoglobin; or at least about 20, 25, or 30 pg/cell of        hemoglobin; or    -   d) phosphatidylserine content of the outer leaflet is less than        30%, 25%, 20%, 15%, 10%, or 5% as measured by Annexin V        staining.

In some embodiments, at least one, e.g., all, of the plurality ofexogenous polypeptides are glycosylated. In some embodiments, at leastone, e.g., all, of the plurality of exogenous polypeptides arephosphorylated.

In some embodiments, the enucleated red blood cell is a reticulocyte.

In some embodiments, the exogenous polypeptide or polypeptides lack asortase transfer signature (i.e., a sequence that can be created by asortase reaction) such as LPXTG (SEQ ID NO: 17).

In some aspects, the present disclosure provides a method of treating adisease or condition described herein, comprising administering to asubject in need thereof an enucleated red blood cell, e.g., areticulocyte, described herein. In some embodiments, the disease orcondition is a cancer, e.g., a cancer described herein.

In some aspects, the present disclosure provides a method of bringinginto proximity a first and a second cell surface moiety, e.g.,transmembrane receptors, comprising administering to a subject in needthereof an enucleated red blood cell, e.g., a reticulocyte, describedherein.

In some aspects, the present disclosure provides a method of delivering,presenting, or expressing a plurality of proximity-dependent moleculescomprising providing an enucleated red blood cell, e.g., a reticulocyte,described herein.

In some aspects, the present disclosure provides a method of producingan enucleated red blood cell, e.g., a reticulocyte, described herein,providing contacting a red blood cell precursor with one or more nucleicacids encoding the exogenous polypeptides and placing the cell inconditions that allow enucleation to occur.

In some aspects, the present disclosure provides a preparation, e.g.,pharmaceutical preparation, comprising a plurality of enucleated redblood cells, e.g., reticulocytes, described herein, e.g., at least 10⁸,10⁹, 10¹⁰, 10¹¹, or 10¹² cells.

In some aspects, the present disclosure provides a cell complex, e.g.,an in vitro or in vivo complex, of an engineered red blood cell (RBC),e.g., an enucleated red blood cell, e.g., a reticulocyte, and a targetcell, the complex mediated by one of the exogenous polypeptides. In someembodiments, the cell complex comprises at least 2, 3, 4, 5, 10, 20, 50,or 100 cells. In some embodiments, the cell complex comprises at leasttwo cell types in addition to the engineered RBC, e.g., a cancer celland an immune effector cell.

In some aspects, the present disclosure proves a reaction mixturecomprising an engineered RBC, e.g., an enucleated red blood cell, e.g.,a reticulocyte, and nucleic acid, e.g., one or more nucleic acidmolecules, encoding a multimodal pair described herein. In someembodiments, the nucleic acid comprises at least one promoter that isactive in a red blood cell. In some embodiments, nucleic acid encodes atleast two proteins described herein (e.g., in Table 1, Table 2, andTable 3). In some embodiments, the nucleic acid encodes a thirdexogenous polypeptide.

In some aspects, the present disclosure comprises a method of making anengineered RBC (e.g., an enucleated red blood cell, e.g., areticulocyte) described herein, comprising: providing, e.g., receiving,information about a target cell or subject, responsive to thatinformation selecting a plurality of exogenous polypeptides, andintroducing nucleic acids encoding the exogenous polypeptides into a RBCor RBC precursor.

In some aspects, the present invention comprises a method of evaluatingan engineered erythroid cell, e.g., RBC (e.g., enucleated RBC, e.g., areticulocyte), comprising providing a candidate erythroid cell, e.g.,RBC, and determining if nucleic acid encoding a plurality of exogenouspolypeptides, e.g., a multimodal pair of the exogenous polypeptides, arepresent.

In some aspects, the present invention comprises a method of evaluatingan engineered erythroid cell, e.g., RBC (e.g., enucleated RBC, e.g., areticulocyte), comprising providing a candidate erythroid cell, e.g.,RBC, and determining if a plurality of exogenous polypeptides, e.g., amultimodal pair of exogenous polypeptides, are present, e.g., by proteindetection.

The present disclosure provides, in some aspects, an enucleatederythroid cell comprising:

a first exogenous polypeptide that interacts with a target, and

a second exogenous polypeptide that modifies the target;

wherein one or more of:

(a) the second exogenous polypeptide comprises a moiety that cleaves anantibody, e.g., that cleaves at a hinge region, a CH2 region, or betweena hinge and CH2 region, e.g., an IdeS polypeptide;

(b) the second exogenous polypeptide comprises an enzyme (e.g., aprotease) that modifies, e.g., is specific, e.g., binds to a site ontarget, binds (e.g., specifically) and modifies, e.g., covalentlymodifies, e.g., cleaves, or removes or attaches a moiety to, the target;

(c) the second exogenous polypeptide comprises a polypeptide, e.g., anenzyme, e.g., a protease, that modifies the secondary, tertiary, orquaternary structure of the target, and, in embodiments, alters, e.g.,decreases or increases, the ability of the target to interact withanother molecule, e.g., the first exogenous polypeptide or a moleculeother than the first exogenous polypeptide, wherein optionally thetarget comprises an antibody, or complement factor;

(d) the second exogenous polypeptide comprises a polypeptide, e.g., anenzyme (e.g., a protease) that cleaves the target, e.g., a polypeptide,between a first target domain and a second target domain, e.g., a firsttarget domain that binds a first substrate and a second target domainthat binds a second substrate;

(e) the target is a polypeptide, a carbohydrate (e.g., a glycan), alipid (e.g., a phospholipid), or a nucleic acid (e.g., DNA, or RNA);

(f) the first exogenous polypeptide binds a target, e.g., an antibody,but does not cleave, and the second exogenous polypeptide cleaves a bonde.g., a covalent bond, e.g., a covalent bond in the antibody;

(g) the target comprises an antibody (e.g., an anti-drug antibody) andthe first exogenous polypeptide binds the variable region of theantibody target;

(h) the target comprises an antibody (e.g., an anti-drug antibody) andfirst exogenous polypeptide binds the constant region of the antibodytarget;

(i) the first exogenous polypeptide has an affinity for the target thatis about 1-2 pM, 2-5 pM, 5-10 pM, 10-20 pM, 20-50 pM, 50-100 pM, 100-200pM, 200-500 pM, 500-1000 pM, 1-2 nM, 2-5 nM, 5-10 nM, 10-20 nM, 20-50nM, 50-100 nM, 100-200 nM, 200-500 nM, 500-1000 nM, 1-2 μM, 2-5 μM, 5-10μM, 10-20 μM, 20-50 μM, or 50-100 μM;

(j) the second exogenous polypeptide has a K_(M) for the target of about10⁻¹-10⁻⁷M, 10⁻¹-10⁻²M, 10⁻²-10⁻³M, 10⁻³-10⁻⁴M, 10⁻⁴-10⁻⁵M, 10⁻⁵-10⁻⁶M,or 10⁻⁶-10⁻⁷M;

(k) a ratio of the K_(d) of the first exogenous polypeptide for thetarget (measured in M) divided by the K_(M) of the second exogenouspolypeptide for the target (measured in M) is about 1×10⁻⁹-2×10⁻⁹,2×10⁻⁹-5×10⁻⁹, 5×10⁻⁹-1×10⁻⁸, 1×10⁻⁸-2×10⁻⁸, 2×10⁻⁸-5×10⁻⁸,5×10⁻⁸-1×10⁻⁷, 1×10⁻⁷-2×10⁻⁷, 2×10⁻⁷-5×10⁻⁷, 5×10⁻⁷-1×10⁻⁶,1×10⁻⁶-2×10⁻⁶, 2×10⁻⁶-5×10⁻⁶, 5×10⁻⁶-1×10⁻⁵, 1×10⁻⁵-2×10⁻⁵,2×10⁻⁵-5×10⁻⁵, 5×10⁻⁵-1×10⁻⁴, 1×10⁻⁴-2×10⁻⁴, 2×10⁻⁴-5×10⁻⁴,5×10⁻⁴-1×10⁻³, 1×10⁻³-2×10⁻³, 2×10⁻³-5×10⁻³, 5×10⁻³-1×10⁻²,1×10⁻²-2×10⁻², 2×10⁻²-5×10⁻², 5×10⁻²-1×10⁻¹, 1×10⁻¹-2×10⁻¹,2×10⁻¹-5×10⁻¹, or 5×10⁻¹-1;

(l) the observed reaction rate of the second exogenous polypeptidemodifying the target is greater than the reaction rate of an enucleatedcell which is similar but which lacks the first exogenous polypeptideunder otherwise similar reaction conditions;

(m) a ratio of the average number of the first exogenous polypeptide onthe erythroid cell to the average number of the second exogenouspolypeptide on the erythroid cell is about 50:1, 20:1, 10:1, 8:1, 6:1,4:1, 2:1, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, 1:20, or 1:50;

(n) affinity of the first exogenous polypeptide for the target isgreater than the affinity of the first exogenous polypeptide for themodified (e.g., cleaved) target;

(o) a therapeutically effective dose of the enucleated erythroid cell isless than stoichiometry (e.g., less by 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 99.99%) to the amount oftarget in a subject's peripheral blood at the time of administration;

(p) the number of enucleated erythroid cells in an effective dose, isless than (e.g., less by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, 99.5%, 99.9%, or 99.99%) the number of targets, e.g.,target molecules, in the subject's peripheral blood at the time ofadministration;

(q) the number of second exogenous polypeptides comprised by apreselected amount of enucleated erythroid cells, e.g., an effectivedose, or in vitro effective amount of enucleated erythroid cells, isless than (e.g., less by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, 99.5%, 99.9%, or 99.99%) a reference value for targets,e.g., less than the number of targets in the peripheral blood of thesubject at the time of administration;

(r) the number of first exogenous polypeptides comprised by apreselected amount of enucleated erythroid cells, e.g., an effectivedose, or in vitro effective amount of enucleated erythroid cells, isless than (e.g., less by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, 99.5%, 99.9%, or 99.99%) a reference value for targets,e.g., less than the number of targets in the peripheral blood of thesubject at the time of administration;

(s) the number of first exogenous polypeptides and the number of secondexogenous polypeptides comprised by a preselected amount of enucleatederythroid cells, e.g., an effective dose, enucleated erythroid cells, iseach less than a reference value for targets, e.g., less than the numberof targets in the peripheral blood of the subject at the time ofadministration;

(t) the second exogenous polypeptide modifies (e.g. cleaves) the targetwith a K_(M) of at least 10⁻¹ M, 10⁻²M, 10⁻³ M, 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶M,or 10⁻⁷ M;

(u) the second exogenous polypeptide comprises a chaperone;

(v) the first exogenous polypeptide comprises a surface-exposed portionand the second exogenous polypeptide comprises a surface exposedportion; or

(w) an effective amount of the enucleated erythroid cells is less than(e.g., less by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%,99%, 99.5%, 99.9%, or 99.99%) an effective amount of otherwise similarenucleated erythroid cells that lack the second exogenous polypeptide.

In embodiments, (b) the second exogenous polypeptide comprises an enzyme(e.g., a protease) that modifies, e.g., is specific, e.g., binds to asite on target, binds (e.g., specifically) and modifies, e.g.,covalently modifies, e.g., cleaves, or removes or attaches a moiety to,the target, wherein the target is optionally an antibody, e.g., ananti-drug antibody. In embodiments the modification alters, e.g.,increases or decreases, the ability of the target to interact withanother molecule, e.g., the first exogenous polypeptide or a moleculeother than the first exogenous polypeptide.

In embodiments, (d) the second exogenous polypeptide comprises apolypeptide, e.g., an enzyme (e.g., a protease) that cleaves the target,e.g., a polypeptide, between a first target domain and a second targetdomain, e.g., a first target domain that binds a first substrate and asecond target domain that binds a second substrate. In embodiments thefirst target domain is released from the second target domain. Inembodiments cleavage alters the affinity one or both of the first targetdomain for a first substrate and the affinity of the second targetdomain for a second substrate. In an embodiment the target comprises anantibody and the first target domain comprises one or more CDRs and thesecond target domain comprises a portion of the constant region, e.g., aFc region.

In embodiments, at least two (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10)of (a)-(w) are present. In embodiments, at least (a) and (e) arepresent. In embodiments, at least (a) and (i) are present. Inembodiments, at least (a) and (j) are present. In embodiments, at least(a) and (m) are present. In embodiments, at least (a) and (q) arepresent. In embodiments, at least (a) and (r) are present. Inembodiments, at least (a) and (s) are present. In embodiments, at least(e) and (i) are present. In embodiments, at least (e) and (j) arepresent. In embodiments, at least (e) and (m) are present. Inembodiments, at least (e) and (q) are present. In embodiments, at least(e) and (r) are present. In embodiments, at least (e) and (s) arepresent. In embodiments, at least (i) and (j) are present. Inembodiments, at least (i) and (m) are present. In embodiments, at least(i) and (q) are present. In embodiments, at least (i) and (r) arepresent. In embodiments, at least (i) and (s) are present. Inembodiments, at least (j) and (m) are present. In embodiments, at least(j) and (q) are present. In embodiments, at least (j) and (r) arepresent. In embodiments, at least (j) and (s) are present. Inembodiments, at least (m) and (q) are present. In embodiments, at least(m) and (r) are present. In embodiments, at least (m) and (s) arepresent. In embodiments, at least (q) and (r) are present. Inembodiments, at least (q) and (s) are present. In embodiments, at least(r) and (s) are present.

In embodiments, at least:

(a) and (b), (a) and (c), (a) and (d), (a) and (e), (a) and (f), (a) and(g), (a) and (h), (a) and (i), (a) and (j), (a) and (k), (a) and (l),(a) and (m), (a) and (n), (a) and (o), (a) and (p), (a) and (q), (a) and(r), (a) and (s), (a) and (t), (a) and (u), (a) and (v), (a) and (w),

(b) and (c), (b) and (d), (b) and (e), (b) and (f), (b) and (g), (b) and(h), (b) and (i), (b) and (j), (b) and (k), (b) and (l), (b) and (m),(b) and (n), (b) and (o), (b) and (p), (b) and (q), (b) and (r), (b) and(s), (b) and (t), (b) and (u), (b) and (v), (b) and (w),

(c) and (d), (c) and (e), (c) and (f), (c) and (g), (c) and (h), (c) and(i), (c) and (j), (c) and (k), (c) and (l), (c) and (m), (c) and (n),(c) and (o), (c) and (p), (c) and (q), (c) and (r), (c) and (s), (c) and(t), (c) and (u), (c) and (v), (c) and (w),

(d) and (e), (d) and (f), (d) and (g), (d) and (h), (d) and (i), (d) and(j), (d) and (k), (d) and (l), (d) and (m), (d) and (n), (d) and (o),(d) and (p), (d) and (q), (d) and (r), (d) and (s), (d) and (t), (d) and(u), (d) and (v), (d) and (w),

(e) and (f), (e) and (g), (e) and (h), (e) and (i), (e) and (j), (e) and(k), (e) and (l), (e) and (m), (e) and (n), (e) and (o), (e) and (p),(e) and (q), (e) and (r), (e) and (s), (e) and (t), (e) and (u), (e) and(v), (e) and (w),

(f) and (g), (f) and (h), (f) and (i), (f) and (j), (f) and (k), (f) and(l), (f) and (m), (f) and (n), (f) and (o), (f) and (p), (f) and (q),(f) and (r), (f) and (s), (f) and (t), (f) and (u), (f) and (v), (f) and(w),

(g) and (h), (g) and (i), (g) and (j), (g) and (k), (g) and (l), (g) and(m), (g) and (n), (g) and (o), (g) and (p), (g) and (q), (g) and (r),(g) and (s), (g) and (t), (g) and (u), (g) and (v), (g) and (w),

(h) and (i), (h) and (j), (h) and (k), (h) and (l), (h) and (m), (h) and(n), (h) and (o), (h) and (p), (h) and (q), (h) and (r), (h) and (s),(h) and (t), (h) and (u), (h) and (v), (h) and (w),

(i) and (j), (i) and (k), (i) and (l), (i) and (m), (i) and (n), (i) and(o), (i) and (p), (i) and (q), (i) and (r), (i) and (s), (i) and (t),(i) and (u), (i) and (v), (i) and (w),

(j) and (k), (j) and (l), (j) and (m), (j) and (n), (j) and (o), (j) and(p), (j) and (q), (j) and (r), (j) and (s), (j) and (t), (j) and (u),(j) and (v), (j) and (w),

(k) and (l), (k) and (m), (k) and (n), (k) and (o), (k) and (p), (k) and(q), (k) and (r), (k) and (s), (k) and (t), (k) and (u), (k) and (v),(k) and (w),

(l) and (m), (l) and (n), (l) and (o), (l) and (p), (l) and (q), (l) and(r), (l) and (s), (l) and (t), (l) and (u), (l) and (v), (l) and (w),

(m) and (n), (m) and (o), (m) and (p), (m) and (q), (m) and (r), (m) and(s), (m) and (t), (m) and (u), (m) and (v), (m) and (w),

(n) and (o), (n) and (p), (n) and (q), (n) and (r), (n) and (s), (n) and(t), (n) and (u), (n) and (v), (n) and (w),

(o) and (p), (o) and (q), (o) and (r), (o) and (s), (o) and (t), (o) and(u), (o) and (v), (o) and (w),

(p) and (q), (p) and (r), (p) and (s), (p) and (t), (p) and (u), (p) and(v), (p) and (w),

(q) and (r), (q) and (s), (q) and (t), (q) and (u), (q) and (v), (q) and(w),

(r) and (s), (r) and (t), (r) and (u), (r) and (v), (r) and (w),

(s) and (t), (s) and (u), (s) and (v), (s) and (w),

(t and (u), (t) and (v), (t) and (w),

(u) and (v), (u) and (w), or

(v) and (w), are present.

In embodiments, the target is other than an infectious component, e.g.,other than a bacterial component, a viral component, a fungal component,or a parasitic component. In embodiments, the first exogenouspolypeptide comprises a target-binding domain. In embodiments, thesurface-exposed portion of the first exogenous polypeptide binds thetarget. In embodiments, the surface-exposed portion of the secondexogenous polypeptide comprises enzymatic activity, e.g., proteaseactivity. In embodiments, the surface-exposed portion of the secondexogenous polypeptide enzymatically modifies, e.g., cleaves, the target.In embodiments, the target comprises an anti-drug antibody, the firstexogenous polypeptide comprises a polypeptide to which the anti-drugantibody binds, and the second exogenous polypeptide comprises aprotease that cleaves the anti-drug antibody to produce a Fab portionand an Fc portion. In embodiments, the enucleated red blood cell iscapable of clearing the target from a subject's body at a faster ratethan an otherwise similar enucleated red blood cell that lacks thesecond exogenous polypeptide. In embodiments, the enucleated red bloodcell is complexed with the target or a reaction product of the secondexogenous protein acting on the target, e.g., during cleavage.

The present disclosure also provides, in certain aspects, an enucleatederythroid cell comprising:

a first exogenous polypeptide comprising a transmembrane domain and asurface-exposed polypeptide capable of binding an anti-drug antibody,and

a second exogenous polypeptide comprising a transmembrane domain and asurface-exposed IdeS polypeptide.

The present disclosure also provides, in some aspects, a polypeptidecomprising a protease that can cleave an antibody, e.g., an IdeSpolypeptide, and a membrane anchor domain, e.g., a transmembrane domain,e.g., type I or type II red blood cell transmembrane domain. Thedisclosure also provides a nucleic acid encoding said polypeptide.

The present disclosure also provides, in some aspects, a nucleic acidcomprising:

a first sequence encoding a protease that can cleave an antibody, e.g.,an IdeS polypeptide,

a second sequence encoding a membrane anchor domain, e.g., atransmembrane domain, wherein the first and second sequences areoperatively linked to form a fusion protein; and

optionally, a promoter sequence that is active in an erythroid cell.

The present disclosure also provides, in some aspects, a nucleic acidcomposition comprising:

a first nucleic acid sequence encoding a first exogenous polypeptidethat interacts with a target, e.g., a first exogenous polypeptidedescribed herein,

a second nucleic acid sequence encoding a second exogenous polypeptidethat modifies the target, e.g., a second nucleic acid sequence describedherein and

optionally, a promoter sequence that is active in an erythroid cell.

In embodiments, the first nucleic acid sequence and second nucleic acidsequence are contiguous or are separate molecules (e.g., admixedmolecules or in separate containers). In embodiments, the first nucleicacid sequence and second nucleic acid sequence are part of the same openreading frame and have a protease cleavage site situated therebetween.In embodiments, the first nucleic acid is operatively linked to a firstpromoter and the second nucleic acid is operatively linked to a secondpromoter.

The disclosure provides, in some aspects, a kit comprising:

(A) nucleic acids encoding: (A-i) a plurality of binding moieties (e.g.,antibody molecules, e.g., scFv domains), fused to (A-ii) a membraneanchor domain, e.g., a transmembrane domain, wherein (A-i) and (A-ii)are operatively linked to a nucleic acid that directs expression in anerythroid cell; and

(B) nucleic acids encoding (B-i) a plurality of enzymes (e.g.,proteases), optionally fused to (B-ii) a membrane anchor domain, e.g., atransmembrane domain, wherein (B-i) and (B-ii) are operatively linked tonucleic acid that directs expression in an erythroid cell.

The present disclosure provides, in some aspects, a method of making afragment of a target, e.g., a target polypeptide, e.g., a method ofmaking a fragment of a target comprising a first target domain, e.g., amethod of making a variable region fragment, or a method of making aconstant region containing fragment, comprising contacting the targetpolypeptide with an erythroid cell described herein. In embodiments, thesecond exogenous polypeptide cleaves the target to provide the fragment.In embodiments, the target polypeptide is an antibody, e.g., ananti-drug antibody. In embodiments, the fragment of the targetpolypeptide does not activate an immune response and/or inflammation. Inembodiments, the contacting comprises administering the erythroid cellto a subject that comprises the target polypeptide.

The present disclosure also provides, in certain aspects, a method ofmaking an inhibitor, e.g., a competitive inhibitor, comprising, e.g.,contacting a precursor of the inhibitor (a target) with an erythroidcell described herein. In embodiments, the second exogenous polypeptideinteracts with the target, e.g., cleaves the target. In embodiments, theinhibitor is an antibody fragment (e.g., a Fab fragment). Inembodiments, the target is an antibody which is cleaved to produce aninhibitor which is an antibody fragment, e.g., Fab fragment. Inembodiments, the inhibitor does not activate an immune response and/orinflammation. In embodiments, the precursor of the inhibitor is anantibody, e.g., an anti-drug antibody. In embodiments, the contactingcomprises administering the erythroid cell to a subject that comprisesthe precursor of the inhibitor.

The present disclosure also provides, in some aspects, a method ofconverting or activating a target, e.g., a polypeptide, e.g., convertinga prodrug to a drug, comprising contacting the polypeptide with anerythroid cell described herein. In embodiments, the second exogenouspolypeptide interacts with the target (e.g., prodrug), e.g., cleaves thetarget. In embodiments, the prodrug is an antibody, e.g., an anti-drugantibody. In embodiments, the drug is an antibody fragment, e.g., a Fabfragment. In embodiments, the drug does not activate an immune responseand/or inflammation. In embodiments, the contacting comprisesadministering the erythroid cell to a subject that comprises thepolypeptide, e.g., prodrug.

The present disclosure also provides, in some aspects, a method ofconverting an endogenous polypeptide from a first activity state to asecond activity state (e.g., from an inactive state to an active stateor an active state to an inactive state), comprising contacting theendogenous polypeptide with an erythroid cell described herein. Inembodiments, the second exogenous polypeptide interacts with the target,e.g., covalently modifies, e.g., cleaves the target, or alters itsability to interact with, e.g., bind, another molecule. In embodiments,the endogenous polypeptide is an antibody, e.g., an anti-drug antibody.In embodiments, the contacting comprises administering the erythroidcell to a subject that comprises the endogenous polypeptide.

The disclosure provides, in some aspects, a method of reducing a levelof a target (e.g., an antibody, e.g., an anti-drug antibody) in asubject, comprising administering to the subject an erythroid celldescribed herein. In embodiments, the second exogenous polypeptideinteracts with the target, e.g., covalently modifies, e.g., cleaves thetarget, or alters its ability to interact with, e.g., bind, anothermolecule. The disclosure also provides, in certain aspects, a method ofgenerating an inhibitory fragment of an antibody (e.g., a Fab fragment)in a subject, comprising administering to the subject an erythrocytecell described herein. The disclosure provides, in addition, a method oftreating a disease in a subject, e.g., cancer, comprising administeringto the subject an erythroid cell described herein.

In embodiments, e.g., embodiments of any of the methods described above,the erythroid cell comprises:

a first exogenous polypeptide that interacts with a target, and

a second exogenous polypeptide that modifies the target;

wherein one or more of:

(a) the second exogenous polypeptide comprises a moiety that cleaves anantibody, e.g., that cleaves at a hinge region, a CH2 region, or betweena hinge and CH2 region, e.g., an IdeS polypeptide;

(b) the second exogenous polypeptide comprises an enzyme (e.g., aprotease) that modifies, e.g., is specific, e.g., binds to a site ontarget, binds (e.g., specifically) and modifies, e.g., covalentlymodifies, e.g., cleaves, or removes or attaches a moiety to, the target,wherein the target is optionally an antibody;

(c) the second exogenous polypeptide comprises a polypeptide, e.g., anenzyme, e.g., a protease, that modifies the secondary, tertiary, orquaternary structure of the target, and, in embodiments, alters, e.g.,decreases or increases, the ability of the target to interact withanother molecule, e.g., the first exogenous polypeptide or a moleculeother than the first exogenous polypeptide, wherein optionally thetarget comprises an antibody, or complement factor;

(d) the second exogenous polypeptide comprises a polypeptide, e.g., anenzyme (e.g., a protease) that cleaves the target, e.g., a polypeptide,between a first target domain and a second target domain, e.g., a firsttarget domain that binds a first substrate and a second target domainthat binds a second substrate;

(e) the target is a polypeptide, a carbohydrate (e.g., a glycan), alipid (e.g., a phospholipid), or a nucleic acid (e.g., DNA or RNA);

(f) the first exogenous polypeptide binds a target, e.g., an antibody,but does not cleave, and the second exogenous polypeptide cleaves a bonde.g., a covalent bond, e.g., a covalent bond in the antibody;

(g) the target comprises an antibody and the first exogenous polypeptidebinds the variable region of the antibody target;

(h) the target comprises an antibody and first exogenous polypeptidebinds the constant region of the antibody target;

(i) the first exogenous polypeptide has an affinity for the target thatis about 1-2 pM, 2-5 pM, 5-10 pM, 10-20 pM, 20-50 pM, 50-100 pM, 100-200pM, 200-500 pM, 500-1000 pM, 1-2 nM, 2-5 nM, 5-10 nM, 10-20 nM, 20-50nM, 50-100 nM, 100-200 nM, 200-500 nM, 500-1000 nM, 1-2 μM, 2-5 μM, 5-10μM, 10-20 μM, 20-50 μM, or 50-100 μM;

(j) the second exogenous polypeptide has a K_(M) for the target of about10⁻¹-10⁻⁷M, 10⁻¹-10⁻²M, 10⁻²-10⁻³M, 10⁻³-10⁻⁴M, 10⁻⁴-10⁻⁵M, 10⁻⁵-10⁻⁶M,or 10⁻⁶-10⁻⁷M;

(k) a ratio of the K_(d) of the first exogenous polypeptide for thetarget (measured in M) divided by the K_(M) of the second exogenouspolypeptide for the target (measured in M) is about 1×10⁻⁹-2×10⁻⁹,2×10⁻⁹-5×10⁻⁹, 5×10⁻⁹-1×10⁻⁸, 1×10⁻⁸-2×10⁻⁸, 2×10⁻⁸-5×10⁻⁸,5×10⁸-1×10⁻⁷, 1×10⁻⁷-2×10⁻⁷, 2×10⁻⁷-5×10⁻⁷, 5×10⁻⁷-1×10⁻⁶,1×10⁻⁶-2×10⁻⁶, 2×10⁻⁶-5×10⁻⁶, 5×10⁻⁶-1×10⁻⁵, 1×10⁻⁵-2×10⁻⁵,2×10⁻⁵-5×10⁻⁵, 5×10⁻⁵-1×10⁻⁴, 1×10⁻⁴-2×10⁻⁴, 2×10⁻⁴-5×10⁻⁴,5×10⁻⁴-1×10⁻³, 1×10⁻³-2×10⁻³, 2×10⁻³-5×10⁻³, 5×10⁻³-1×10⁻²,1×10⁻²-2×10⁻², 2×10⁻²-5×10⁻², 5×10⁻²-1×10⁻¹, 1×10⁻¹-2×10⁻¹,2×10⁻¹-5×10⁻¹, or 5×10⁻¹-1;

(l) the observed reaction rate of the second exogenous polypeptidemodifying the target is greater than the reaction rate of an enucleatedcell which is similar but which lacks the first exogenous polypeptideunder otherwise similar reaction conditions;

(m) a ratio of the average number of the first exogenous polypeptide onthe erythroid cell to the average number of the second exogenouspolypeptide on the erythroid cell is about 50:1, 20:1, 10:1, 8:1, 6:1,4:1, 2:1, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, 1:20, or 1:50;

(n) affinity of the first exogenous polypeptide for the target isgreater than the affinity of the first exogenous polypeptide for themodified (e.g., cleaved) target;

(o) a therapeutically effective dose of the enucleated erythroid cell isless than (e.g., less by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, 99.5%, 99.9%, or 99.99%) stoichiometry to the amount oftarget in a subject's peripheral blood at the time of administration;

(p) the number of enucleated erythroid cells in an effective dose, isless than (e.g., less by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, 99.5%, 99.9%, or 99.99%) the number of targets, e.g.,target molecules, in the subject's peripheral blood at the time ofadministration;

(q) the number of second exogenous polypeptides comprised by apreselected amount of enucleated erythroid cells, e.g., an effectivedose of enucleated erythroid cells, is less than (e.g., less by 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or99.99%) a reference value for targets, e.g., less than the number oftargets in the peripheral blood of the subject at the time ofadministration;

(r) the number of first exogenous polypeptides comprised by apreselected amount of enucleated erythroid cells, e.g., an effectivedose of enucleated erythroid cells, is less than (e.g., less by 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or99.99%) a reference value for targets, e.g., less than the number oftargets in the peripheral blood of the subject at the time ofadministration;

(s) the number of first exogenous polypeptides and the number of secondexogenous polypeptides comprised by a preselected amount of enucleatederythroid cells, e.g., an effective dose, enucleated erythroid cells, iseach less than (e.g., less by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, 99.5%, 99.9%, or 99.99%) a reference value fortargets, e.g., less than the number of targets in the peripheral bloodof the subject at the time of administration;

(t) the second exogenous polypeptide modifies (e.g. cleaves) the targetwith a K_(M) of at least 10⁻¹ M, 10⁻²M, 10⁻³ M, 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶M,or 10⁻⁷ M;

(u) the second exogenous polypeptide comprises a chaperone;

(v) the first exogenous polypeptide comprises a surface-exposed portionand the second exogenous polypeptide comprises a surface exposedportion; or

(w) an effective amount of the enucleated erythroid cells is less than(e.g., less by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%,99%, 99.5%, 99.9%, or 99.99%) an effective amount of otherwise similarenucleated erythroid cells that lack the second exogenous polypeptide.

In some embodiments of any of the compositions and methods describedherein involving an exogenous polypeptide, e.g., a fusion protein:

-   -   i) at least 50, 60, 70, 80, 90, 95, or 99% of the fusion        proteins on the surface of the erythroid cell have an identical        sequence,    -   ii) at least 50, 60, 70, 80, 90, 95, or 99% of the fusion        protein have the same transmembrane region,    -   iii) the fusion protein does not include a full length        endogenous membrane protein, e.g., comprises a segment of a full        length endogenous membrane protein, which segment lacks at least        1, 2, 3, 4, 5, 10, 20, 50, 100, 200, or 500 amino acids of the        full length endogenous membrane protein;    -   iv) at least 50, 60, 70, 80, 90, 95, or 99% of the fusion        proteins do not differ from one another by more than 1, 2, 3, 4,        5, 10, 20, or 50 amino acids,    -   v) the exogenous polypeptide lacks a sortase transfer signature,    -   vi) the exogenous polypeptide comprises a moiety that is present        on less than 1, 2, 3, 4, or 5 sequence distinct fusion        polypeptides;    -   vii) the exogenous polypeptide is present as a single fusion        polypeptide;    -   viii) the fusion protein does not contain Gly-Gly at the        junction of an endogenous transmembrane protein and the moiety;    -   ix) the fusion protein does not contain Gly-Gly, or the fusion        protein does not contain Gly-Gly, or does not contain Gly-Gly in        an extracellular region, does not contain Gly-Gly in an        extracellular region that is within 1, 2, 3, 4, 5, 10, 20, 50,        or 100 amino acids of a transmembrane segment; or a combination        thereof.

The cell systems described herein may be used in combination withanother (one or more) anti-proliferative, anti-neoplastic or anti-tumordrug or treatment that is not part of the cell system. Such drugs ortreatments include chemotherapeutic drugs, e.g., cytotoxic drugs (e.g.,alkylating agents, antimetabolites, anti-tumor antibiotics,topoisomerase inhibitors, mitotic inhibitors, corticosteroids); cancergrowth blockers such as tyrosine kinase inhibitors and proteasomeinhibitors; T cell therapy (e.g., CAR-T cell therapy) (see, e.g., PMID:26611350), Natural Killer (NK) cell immunomodulation (see, e.g., PMID:26697006); and cancer vaccines (PMID: 26579225); other chemical drugssuch as L-asparaginase and bortezomib (Velcade®). Hormone therapies (oranti-hormone therapies) may be used, e.g., for hormone-sensitivecancers.

The cell systems described herein may also be used in combination withnon-drug therapies for cancer such as surgery, radiotherapy, orcryotherapy. In some cases, treatment methods of the invention mayinclude a cell system described herein in combination with 2 or moreother therapies or drugs, e.g., breast cancer may be treated with acombination of a cell system described herein in combination withsurgery or radiotherapy and a chemotherapeutic cocktail or biologic(e.g., an anti-HER2 antibody).

The disclosure contemplates all combinations of any one or more of theforegoing aspects and/or embodiments, as well as combinations with anyone or more of the embodiments set forth in the detailed description andexamples.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references (e.g., sequencedatabase reference numbers) mentioned herein are incorporated byreference in their entirety. For example, all GenBank, Unigene, andEntrez sequences referred to herein, e.g., in any Table herein, areincorporated by reference. Unless otherwise specified, the sequenceaccession numbers specified herein, including in any Table herein, referto the database entries current as of Jan. 11, 2016. When one gene orprotein references a plurality of sequence accession numbers, all of thesequence variants are encompassed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of graphs showing results of a Raji apoptosis assaymeasured through flow cytometry. Raji cells are CFSE labeled andco-cultured with erythroid differentiated cells that are untransduced(control) and transduced with single or multiple TRAIL variants orco-cultured with two different singly transduced cells. Percentapoptosis determined by percent of cells that are Raji (CFSE+) andannexin V+. (Top) Flow cytometry plots of CFSE and annexin V staining ofvarious conditions. (Bottom) Graph of percent apoptosis of the variousconditions.

FIG. 2 is a bar graph showing the mean fluorescent intensity fromcontrol erythroid cells (UNT) or IdeS-expressing erythroid cells (IDES)labelled with an anti-Rabbit Fc fluorophore labeled antibody, before orafter a 5 hour incubation.

FIG. 3 is a Western blot showing intact heavy chain of target antibodiesor fragments of the heavy chain in supernatant from control cells (UNT)or Ide-S expressing cells (IdeS-RCT). Arrows indicate the heavy chain(Hc), heavy chain fragment (Hc-fragment), and light chain (Lc).

FIG. 4 is a diagram of an erythroid cell comprising a first exogenouspolypeptide (white), a second exogenous polypeptide (hatching), and athird exogenous polypeptide (close hatching) wherein each exogenouspolypeptide comprises a capture agent capable of trapping a target,e.g., an unwanted target. The erythroid cell can engage in dualtrapping, where it uses more than one exogenous polypeptide to bind asingle or multiple soluble factors.

FIG. 5 is a diagram of an erythroid cell comprising a first exogenouspolypeptide and a second exogenous polypeptide wherein each exogenouspolypeptide is capable of trapping an antibody, e.g., unwanted antibody,e.g., an anti-drug antibody.

FIG. 6 is a diagram of an erythroid cell comprising a first exogenouspolypeptide that binds a target, e.g., an antibody, e.g., an unwantedantibody, e.g., an anti-drug antibody, and a second exogenouspolypeptide that modifies the target, e.g., cleaves the target. Thesecond exogenous polypeptide may comprise a protease such as IdeS.

FIG. 7 is a diagram of an erythroid cell comprising a first exogenouspolypeptide that binds a target, e.g., an unwanted anti-drug antibodyproduced by a subject in reaction to treatment with a drug, a secondexogenous polypeptide that cleaves the target, and an optional thirdexogenous polypeptide comprising a therapeutic protein, e.g., analternative to the drug against which the subject produced anti-drugantibodies.

FIG. 8 is a diagram of an erythroid cell comprising a first exogenouspolypeptide with therapeutic activity (e.g., an anti-CD40 antibodymolecule), a second exogenous polypeptide (e.g., CD40 or a fragment orvariant thereof) that inhibits the first exogenous polypeptide, andoptionally a third exogenous polypeptide that comprises a targetingagent, e.g., an anti-CD20 antibody molecule.

FIG. 9 is a diagram of an erythroid cell comprising a first exogenouspolypeptide with a first targeting agent and a second exogenouspolypeptide with a second targeting agent.

FIG. 10 is a diagram of an erythroid cell comprising an antagonistand/or agonist.

FIG. 11 is a diagram or an erythroid cell comprising a targeting agent(e.g., an anti-CD4 antibody molecule) and an internal payload (e.g.,IDO).

FIG. 12 is a diagram of an erythroid cell comprising a first exogenouspolypeptide comprising a targeting agent and a second exogenouspolypeptide comprising an agonist of a target.

FIG. 13 is a diagram of an erythroid cell comprising a first exogenouspolypeptide comprising a targeting agent (e.g., an anti-BCMA antibodymolecule) and a second exogenous polypeptide comprising a capture agent.

FIG. 14 is a diagram of an erythroid cell comprising a first exogenouspolypeptide comprising a targeting agent and a second exogenouspolypeptide (e.g., TRAIL) that promotes a given activity, e.g.,apoptosis.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “antibody molecule” refers to a protein, e.g.,an immunoglobulin chain or fragment thereof, comprising at least oneimmunoglobulin variable domain sequence. The term “antibody molecule”encompasses antibodies and antibody fragments. In an embodiment, anantibody molecule is a multispecific antibody molecule, e.g., abispecific antibody molecule. Examples of antibody molecules include,but are not limited to, Fab, Fab′, F(ab′)2, Fv fragments, scFv antibodyfragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of theVH and CH1 domains, linear antibodies, single domain antibodies such assdAb (either VL or VH), camelid VHH domains, multi-specific antibodiesformed from antibody fragments such as a bivalent fragment comprisingtwo Fab fragments linked by a disulfide bridge at the hinge region, anisolated epitope binding fragment of an antibody, maxibodies,minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies,v-NAR and bis-scFv.

As used herein, a “combination therapy” or “administered in combination”means that two (or more) different agents or treatments are administeredto a subject as part of a treatment regimen for a particular disease orcondition. The treatment regimen includes the doses and periodicity ofadministration of each agent such that the effects of the separateagents on the subject overlap. In some embodiments, the delivery of thetwo or more agents is simultaneous or concurrent and the agents may beco-formulated. In other embodiments, the two or more agents are notco-formulated and are administered in a sequential manner as part of aprescribed regimen. In some embodiments, administration of two or moreagents or treatments in combination is such that the reduction in asymptom, or other parameter related to the disorder is greater than whatwould be observed with one agent or treatment delivered alone or in theabsence of the other. The effect of the two treatments can be partiallyadditive, wholly additive, or greater than additive (e.g., synergistic).Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, oral routes, intravenous routes, intramuscularroutes, and direct absorption through mucous membrane tissues. Thetherapeutic agents can be administered by the same route or by differentroutes. For example, a first therapeutic agent of the combination may beadministered by intravenous injection while a second therapeutic agentof the combination may be administered orally.

The term “coordinated” or “coordinated manner” means that a plurality ofagents work together to provide a therapeutic benefit. Types ofcoordinated activity include agent-additive, agent-synergistic,multiplicative, independent function, localization-based,proximity-dependent, scaffold-based, multimer-based, and compensatoryactivity. In an embodiment the level of therapeutic benefit conferred bya plurality of exogenous polypeptides delivered in the same enucleatedRBC is greater than would be seen if each of the plurality ofpolypeptides were delivered from different enucleated RBCs.

As used herein, “enucleated” refers to a cell that lacks a nucleus,e.g., a cell that lost its nucleus through differentiation into a maturered blood cell.

As used herein, the term “exogenous polypeptide” refers to a polypeptidethat is not produced by a wild-type cell of that type or is present at alower level in a wild-type cell than in a cell containing the exogenouspolypeptide. In some embodiments, an exogenous polypeptide is apolypeptide encoded by a nucleic acid that was introduced into the cell,which nucleic acid is optionally not retained by the cell.

As used herein, the term “multimodal therapy” refers to a therapy, e.g.,an enucleated red blood cell therapy, that provides a plurality (e.g.,2, 3, 4, or 5 or more) of exogenous agents (e.g., polypeptides) thathave a coordinated function (e.g., agent-additive, agent-synergistic,multiplicative, independent function, localization-based,proximity-dependent, scaffold-based, multimer-based, or compensatoryactivity).

As used herein, the term “pathway” or “biological pathway” refers to aplurality of biological molecules, e.g., polypeptides, that act togetherin a sequential manner. Examples of pathways include signal transductioncascades. In some embodiments, a pathway begins with detection of anextracellular signal and ends with a change in transcription of a targetgene. In some embodiments, a pathway begins with detection of acytoplasmic signal and ends with a change in transcription of a targetgene. A pathway can be linear or branched. If branched, it can have aplurality of inputs (converging), or a plurality of outputs (diverging).

As used herein, a “proximity-dependent” molecule refers to a firstmolecule that has a different, e.g., greater, activity when in proximitywith a second molecule than when alone. In some embodiments, a pair ofproximity-dependent ligands activates a downstream factor more stronglywhen the ligands are in proximity than when they are distant from eachother.

As used herein, “receptor component” refers to a polypeptide thatfunctions as a receptor, by itself or as part of a complex. Thus areceptor component encompasses a polypeptide receptor and a polypeptidethat functions as part of a receptor complex.

The term “synergy” or “synergistic” means a more than additive effect ofa combination of two or more agents (e.g., polypeptides that are part ofan enucleated red blood cell) compared to their individual effects. Incertain embodiments, synergistic activity is a more-than-additive effectof an enucleated red blood cell comprising a first polypeptide and asecond polypeptide, compared to the effect of an enucleated red bloodcell comprising the first polypeptide and an enucleated red blood cellcomprising the second polypeptide. In some embodiments, synergisticactivity is present when a first agent produces a detectable level of anoutput X, a second agent produces a detectable level of the output X,and the first and second agents together produce a more-than-additivelevel of the output X.

As used herein, the term “variant” of a polypeptide refers to apolypeptide having at least one sequence difference compared to thatpolypeptide, e.g., one or more substitutions, insertions, or deletions.In some embodiments, the variant has at least 70%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identity to that polypeptide. A variant includes afragment. In some embodiments, a fragment lacks up to 1, 2, 3, 4, 5, 10,20, or 100 amino acids on the N-terminus, C-terminus, or both (eachindependently), compared to the full-length polypeptide.

Exemplary Exogenous Polypeptides and Uses Thereof

In embodiments, the red blood cell therapeutics described hereincomprise one or more (e.g., 2, 3, 4, 5, 6, 10 or more) differentexogenous agents, e.g., exogenous polypeptides, lipids, or smallmolecules. In some embodiments, a red blood cell therapeutic comprisesan exogenous fusion polypeptide comprising two or more differentproteins described herein. In some embodiments, an enucleated red bloodcell, e.g., a reticulocyte, comprises two or more different exogenouspolypeptides described herein. In some embodiments, one or more (e.g.,all) of the exogenous polypeptides are human polypeptides or fragmentsor variants thereof.

In some embodiments, the two or more polypeptides act on the sametarget, and in other embodiments, they act on two or more differenttargets. In some embodiments, the single target or plurality of targetsis chosen from an endogenous human protein or a soluble factor (e.g., apolypeptide, small molecule, or cell-free nucleic acid).

One or more of the exogenous proteins may have post-translationalmodifications characteristic of eukaryotic cells, e.g., mammalian cells,e.g., human cells. In some embodiments, one or more (e.g., 2, 3, 4, 5,or more) of the exogenous proteins are glycosylated, phosphorylated, orboth. In vitro detection of glycoproteins is routinely accomplished onSDS-PAGE gels and Western Blots using a modification of Periodicacid-Schiff (PAS) methods. Cellular localization of glycoproteins may beaccomplished utilizing lectin fluorescent conjugates known in the art.Phosphorylation may be assessed by Western blot using phospho-specificantibodies.

Post-translation modifications also include conjugation to a hydrophobicgroup (e.g., myristoylation, palmitoylation, isoprenylation,prenylation, or glypiation), conjugation to a cofactor (e.g.,lipoylation, flavin moiety (e.g., FMN or FAD), heme C attachment,phosphopantetheinylation, or retinylidene Schiff base formation),diphthamide formation, ethanolamine phosphoglycerol attachment, hypusineformation, acylation (e.g. O-acylation, N-acylation, or S-acylation),formylation, acetylation, alkylation (e.g., methylation or ethylation),amidation, butyrylation, gamma-carboxylation, malonylation,hydroxylation, iodination, nucleotide addition such as ADP-ribosylation,oxidation, phosphate ester (O-linked) or phosphoramidate (N-linked)formation, (e.g., phosphorylation or adenylylation), propionylation,pyroglutamate formation, S-glutathionylation, S-nitrosylation,succinylation, sulfation, ISGylation, SUMOylation, ubiquitination,Neddylation, or a chemical modification of an amino acid (e.g.,citrullination, deamidation, eliminylation, or carbamylation), formationof a disulfide bridge, racemization (e.g., of proline, serine, alanine,or methionine). In embodiments, glycosylation includes the addition of aglycosyl group to arginine, asparagine, cysteine, hydroxylysine, serine,threonine, tyrosine, or tryptophan, resulting in a glycoprotein. Inembodiments, the glycosylation comprises, e.g., O-linked glycosylationor N-linked glycosylation.

In some embodiments, one or more of the exogenous polypeptides is afusion protein, e.g., is a fusion with an endogenous red blood cellprotein or fragment thereof, e.g., a transmembrane protein, e.g., GPA ora transmembrane fragment thereof. In some embodiments, one or more ofthe exogenous polypeptides is fused with a domain that promotesdimerization or multimerization, e.g., with a second fusion exogenouspolypeptide, which optionally comprises a dimerization domain. In someembodiments, the dimerization domain comprises a portion of an antibodymolecule, e.g., an Fc domain or CH3 domain. In some embodiments, thefirst and second dimerization domains comprise knob-in-hole mutations(e.g., a T366Y knob and a Y407T hole) to promote heterodimerization.

An exemplary human polypeptide, e.g., a human polypeptide selected fromany of Tables 1-4, includes:

a) a naturally occurring form of the human polypeptide, e.g., anaturally occurring form of the human polypeptide that is not associatedwith a disease state;

b) the human polypeptide having a sequence appearing in a database,e.g., GenBank database, on Jan. 11, 2017, for example a naturallyoccurring form of the human polypeptide that is not associated with adisease state having a sequence appearing in a database, e.g., GenBankdatabase, on Jan. 11, 2017;

c) a human polypeptide having a sequence that differs by no more than 1,2, 3, 4, 5 or 10 amino acid residues from a sequence of a) or b);

d) a human polypeptide having a sequence that differs at no more than 1,2, 3, 4, 5 or 10% its amino acids residues from a sequence of a) or b);

e) a human polypeptide having a sequence that does not differsubstantially from a sequence of a) or b); or

f) a human polypeptide having a sequence of c), d), or e) that does notdiffer substantially in a biological activity, e.g., an enzymaticactivity (e.g., specificity or turnover) or binding activity (e.g.,binding specificity or affinity) from a human polypeptide having thesequence of a) or b). Candidate peptides under f) can be made andscreened for similar activity as described herein and would beequivalent hereunder if expressed in enucleated RBCs as describedherein).

In embodiments, an exogenous polypeptide comprises a human polypeptideor fragment thereof, e.g., all or a fragment of a human polypeptide ofa), b), c), d), e), or f) of the preceding paragraph. In an embodiment,the exogenous polypeptide comprises a fusion polypeptide comprising allor a fragment of a human polypeptide of a), b), c), d), e), or f) of thepreceding paragraph and additional amino acid sequence. In an embodimentthe additional amino acid sequence comprises all or a fragment of humanpolypeptide of a), b), c), d), e), or f) of the preceding paragraph fora different human polypeptide.

The invention contemplates that functional fragments or variants thereof(e.g., a ligand-binding fragment or variant thereof of the receptorslisted in Tables 1-4) can be made and screened for similar activity asdescribed herein and would be equivalent hereunder if expressed inenucleated RBCs as described herein).

In embodiments, the two or more exogenous agents (e.g., polypeptides)have related functions that are agent-additive, agent-synergistic,multiplicative, independent function, localization-based,proximity-dependent, scaffold-based, multimer-based, or compensatory, asdescribed herein. In some embodiments, more than one of thesedescriptors applies to a given RBC.

Agent-Additive Configurations

When two or more agents (e.g., polypeptides) are agent-additive, theeffect of the agents acting together is greater than the effect ofeither agent acting alone. In an embodiment, two agents have different(e.g., complementary) functions in the RBC (e.g., on the RBC surface)and act together to have a stronger effect (compared to either of theagents acting alone), e.g., a higher binding affinity for the target, ora greater degree of modulation of signal transduction by the target,e.g., a single target. In some embodiments, two or more agents each bindto the same target, e.g., to different epitopes within the same targetprotein.

In an embodiment the agents associate with one another, e.g., aremembers of a heterodimeric complex. In an embodiment, the agents havegreater avidity for a target when acting together than when actingalone.

In some embodiments, the two or more agents enable tighter binding to atarget than either agent alone. In some embodiments, a heterodimer ofreceptor components, e.g., cytokine receptor components, e.g.,interleukin receptor components, e.g., IL-1 receptor components, bind toa target, e.g., IL-1, with higher affinity than either receptorcomponent alone. Many signaling molecules form heterodimers orheteromultimers on the cell surface to bind to their ligand. Cytokinereceptors, for example, can be heterodimers or heteromultimers. Forinstance, IL-2 receptor comprises three different molecules: IL2Ra,IL2Rb, and IL2Rg. The IL-13 receptor is a heterodimer of IL13Ra andIL4R. The IL-23 receptor is a heterodimer of IL23R and IL12Rb1. The TNFareceptor is, in embodiments, a heterodimer of TNFR1 and TNFR2. In someembodiments, one or more of the exogenous polypeptides comprises acytokine of Table 1, or a cytokine receptor-binding fragment or variantthereof. The expressed cytokines typically have the wild type humanreceptor sequence or a variant or fragment thereof that is able to bindand signal through its target receptor. A table of cytokines and theirreceptors is provided herein as Table 1. The cytokines can be present onthe surface of the RBC.

TABLE 1 Cytokines and Receptors Name Cytokine Receptor(s)(Da) and FormInterleukins IL-1-like IL-1α CD121a, CDw121b IL-1β CD121a, CDw121bIL-1RA CD121a IL-18 IL-18Rα, β Common g chain (CD132) IL-2 CD25, 122,132 IL-4 CD124, 213a13, 132 IL-7 CD127, 132 IL-9 IL-9R, CD132 IL-13CD213a1, 213a2, IL-15 IL-15Ra, CD122, 132 Common b chain (CD131) IL-3CD123, CDw131 IL-5 CDw125, 131 Also related GM-CSF CD116, CDw131IL-6-like IL-6 CD126, 130 IL-11 IL-11Ra, CD130 Also related G-CSF CD114IL-12 CD212 LIF LIFR, CD130 OSM OSMR, CD130 IL-10-like IL-10 CDw210IL-20 IL-20Rα, β Others IL-14 IL-14R IL-16 CD4 IL-17 CDw217 InterferonsIFN-α CD118 IFN-β CD118 IFN-γ CDw119 TNF CD154 CD40 LT-β LTβR TNF-αCD120a, b TNF-β (LT-α) CD120a, b 4-1BBL CD137 (4-1BB) APRIL BCMA, TACICD70 CD27 CD153 CD30 CD178 CD95 (Fas) GITRL GITR LIGHT LTbR, HVEM OX40LOX40 TALL-1 BCMA, TACI TRAIL TRAILR1-4 TWEAK Apo3 TRANCE RANK, OPG TGF-βTGF-β1 TGF-βR1 TGF-β2 TGF-βR2 TGF-β3 TGF-βR3 Miscellaneoushematopoietins Epo EpoR Tpo TpoR Flt-3L Flt-3 SCF CD117 M-CSF CD115 MSPCDw136

In some embodiments the agents are different antibody-binding molecules,e.g., Fc-binding molecules, for the capture of antibodies incirculation, e.g., anti-drug antibodies. In embodiments, the agents arenon-competitive with one another to enable higher affinity binding ofindividual antibodies or opsonized particles. For example, inembodiments, one or more agent is chosen from protein A, Fc receptor 1(FcR1), FcR2a, FcR2b, FcR3, FcR4, FcRn (neonatal Fc receptor) or anantibody-binding fragment or variant thereof.

In some embodiments the target is a circulating cancer cell, e.g. acancerous B cell, T cell, lymphoid cell, or a circulating tumor cell(CTC). In embodiments, the engineered red blood cell (e.g., areticulocyte) is used to capture the cancer cell and remove it fromcirculation. For instance, the one or more agents bind to differentproteins on the cell surface to enhance the specificity of the therapy.For example the agents comprise anti-EPCAM and anti CD45 antibodymolecules to capture CTCs, or anti-CD19 and anti-CD20 antibody moleculesto capture B cell lineage acute leukemic cells.

An enucleated erythroid cell can comprise a first exogenous polypeptidethat interacts with a target (e.g., an anti-drug antibody) and a secondexogenous polypeptide (e.g., a protease, e.g., IdeS) that modifies thetarget. In embodiments, the erythroid cell is administered to a subject,e.g., a subject having a cancer, e.g., a cancer described herein.

In embodiments, an effective amount of the enucleated erythroid cellscomprising a first exogenous polypeptide and a second exogenouspolypeptide is less than (e.g., less by 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 99.99%) an effectiveamount of otherwise similar enucleated erythroid cells that lack thefirst exogenous polypeptide or lack the second exogenous polypeptide. Inembodiments, the preselected amount is an effective dose or an in vitroeffective amount of enucleated erythroid cells. In embodiments, thepreselected amount (e.g., in vitro effective amount) is an amount thatis effective in an assay, e.g., to convert at least 10%, 20%, 30%, 405,50%, 60%, 70%, 80%, or 90% of substrate into produce in a preselectedamount of time, e.g., 1, 2, 3, 4, 5, or 6 hours. In embodiments, thepreselected amount (e.g., in vitro effective amount) is effective tocleave at least 50% of a target antibody in 5 hours. The assay maymeasure, e.g., reduction in levels of soluble, unmodified (e.g.,non-cleaved) target in a solution.

In embodiments, the reference value for targets is the number of targetsin the peripheral blood of the subject at the time of administration. Inembodiments (e.g., embodiments involving an in vitro effective amount ofcells) the reference value for targets is the number of targets in areaction mixture for an assay.

First Exogenous Polypeptide (e.g., a Binding Agent)

The first exogenous polypeptide can bind a target. In embodiments, thefirst exogenous polypeptide comprises a binding domain that recognizesan antibody, e.g., an anti-drug antibody.

In embodiments, the first exogenous polypeptide comprises a bindingdomain and a membrane anchor domain (e.g., a transmembrane domain, e.g.,type I or type II red blood cell transmembrane domain). In embodiments,the membrane anchor domain is C-terminal or N-terminal of the modifier(e.g., protease) domain. In embodiments, the transmembrane domaincomprises GPA or a transmembrane portion thereof, e.g., as set out inSEQ ID NO: 9 herein or a transmembrane portion thereof, or a polypeptidehaving at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to any of the foregoing. In embodiments, the GPA polypeptide isC-terminal of the binding domain.

In embodiments, the first exogenous polypeptide comprises an addressmoiety or targeting moiety described in WO2007030708, e.g., in pages34-45 therein, which application is herein incorporated by reference inits entirety.

Other examples of proteins that can be suitably adapted for use as thefirst exogenous polypeptide include ligand binding domains of receptors,such as where the target is the receptor ligand. Conversely, the firstexogenous polypeptide can comprise a receptor ligand where the target isthe receptor. A target ligand can be a polypeptide or a small moleculeligand.

In a further embodiment, a first exogenous polypeptide may comprise adomain derived from a polypeptide that has an immunoglobulin-like fold,such as the 10th type III domain of human fibronectin (“Fn3”). See U.S.Pat. Nos. 6,673,901; 6,462,189. Fn3 is small (about 95 residues),monomeric, soluble and stable. It does not have disulfide bonds whichpermit improved stability in reducing environments. The structure may bedescribed as a beta-sandwich similar to that of Ab VH domain except thatFn3 has seven beta-strands instead of nine. There are three loops oneach end of Fn3; and the positions of three of these loops correspond tothose of CDR1, 2 and 3 of the VH domain. The 94 amino acid Fn3 sequenceis:

(SEQ ID NO: 18) VSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITGYAVTGRGDSPASSKPISINYRT

The amino acid positions of the CDR-like loops will be defined asresidues 23-30 (BC Loop), 52-56 (DE Loop) and 77-87 (FG Loop).Accordingly, one or more of the CDR-like loops may be modified orrandomized, to generate a library of Fn3 binding domains which may thenbe screened for binding to a desired address binding site. See also PCTPublication WO0232925. Fn3 is an example of a large subfamily of theimmunoglobulin superfamily (IgSF). The Fn3 family includes cell adhesionmolecules, cell surface hormone and cytokine receptors, chaperonin, andcarbohydrate-binding domains, all of which may also be adapted for useas binding agents. Additionally, the structure of the DNA bindingdomains of the transcription factor NF-kB is also closely related to theFn3 fold and may also be adapted for use as a binding agent. Similarly,serum albumin, such as human serum albumin contains animmunoglobulin-like fold that can be adapted for use as a targetingmoiety.

In still other embodiments, the first exogenous polypeptide can comprisean engineered polypeptide sequence that was selected, e.g.,synthetically evolved, based on its kinetics and selectivity for bindingto the address site. In embodiments, the sequence of the first exogenouspolypeptide is designed using a screen or selection method, e.g., byphage display or yeast two-hybrid screen.

In some embodiments, the first exogenous polypeptide comprises a peptideligand for a soluble receptor (and optionally the target comprises asoluble receptor), a synthetic peptide that binds a target, a complementregulatory domain (and optionally the target comprises a complementfactor), or a ligand for a cell surface receptor (and optionally thetarget comprises the cell surface receptor).

Second Exogenous Polypeptide (e.g., Protease)

In embodiments, the second exogenous polypeptide (which modifies thetarget) is a factor set out in Table 4. In some embodiments, theprotease is a protease set out in Table 4. In embodiments, the proteaseis a bacterial protease, a human protease, or a plant protease, or afragment or variant thereof.

In embodiments, the second exogenous polypeptide (which modifies thetarget) is a protease. Exemplary proteases include those classified asAminopeptidases; Dipeptidases; Dipeptidyl-peptidases and tripeptidylpeptidases; Peptidyl-dipeptidases; Serine-type carboxypeptidases;Metallocarboxypeptidases; Cysteine-type carboxypeptidases;Omegapeptidases; Serine proteinases; Cysteine proteinases; Asparticproteinases; Metalloproteinases; or Proteinases of unknown mechanism.

Aminopeptidases include cytosol aminopeptidase (leucyl aminopeptidase),membrane alanyl aminopeptidase, cystinyl aminopeptidase, tripeptideaminopeptidase, prolyl aminopeptidase, arginyl aminopeptidase, glutamylaminopeptidase, x-pro aminopeptidase, bacterial leucyl aminopeptidase,thermophilic aminopeptidase, clostridial aminopeptidase, cytosol alanylaminopeptidase, lysyl aminopeptidase, x-trp aminopeptidase, tryptophanylaminopeptidase, methionyl aminopeptidase, d-stereospecificaminopeptidase, and aminopeptidase. Dipeptidases include x-hisdipeptidase, x-arg dipeptidase, x-methyl-his dipeptidase, cys-glydipeptidase, glu-glu dipeptidase, pro-x dipeptidase, x-pro dipeptidase,met-x dipeptidase, non-stereospecific dipeptidase, cytosol non-specificdipeptidase, membrane dipeptidase, and beta-ala-his dipeptidase.Dipeptidyl-peptidases and tripeptidyl peptidases includedipeptidyl-peptidase I, dipeptidyl-peptidase II, dipeptidyl peptidaseIII, dipeptidyl-peptidase IV, dipeptidyl-dipeptidase,tripeptidyl-peptidase I, and tripeptidyl-peptidase II.Peptidyl-dipeptidases include peptidyl-dipeptidase A andpeptidyl-dipeptidase B. Serine-type carboxypeptidases include lysosomalpro-x carboxypeptidase, serine-type D-ala-D-ala carboxypeptidase,carboxypeptidase C, and carboxypeptidase D. Metallocarboxypeptidasesinclude carboxypeptidase A, carboxypeptidase B, lysine(arginine)carboxypeptidase, gly-X carboxypeptidase, alanine carboxypeptidase,muramoylpentapeptide carboxypeptidase, carboxypeptidase H, glutamatecarboxypeptidase, carboxypeptidase M, muramoyltetrapeptidecarboxypeptidase, zinc D-ala-D-ala carboxypeptidase, carboxypeptidaseA2, membrane pro-x carboxypeptidase, tubulinyl-tyr carboxypeptidase, andcarboxypeptidase T. Omegapeptidases include acylaminoacyl-peptidase,peptidyl-glycinamidase, pyroglutamyl-peptidase I,beta-aspartyl-peptidase, pyroglutamyl-peptidase II,n-formylmethionyl-peptidase, pteroylpoly-[gamma]-glutamatecarboxypeptidase, gamma-glu-X carboxypeptidase, and acylmuramoyl-alapeptidase. Serine proteinases include chymotrypsin, chymotrypsin C,metridin, trypsin, thrombin, coagulation factor Xa, plasmin,enteropeptidase, acrosin, alpha-lytic protease, glutamyl, endopeptidase,cathepsin G, coagulation factor VIIa, coagulation factor IXa, cucumisi,prolyl oligopeptidase, coagulation factor XIa, brachyurin, plasmakallikrein, tissue kallikrein, pancreatic elastase, leukocyte elastase,coagulation factor XIIa, chymase, complement component clr55, complementcomponent cls55, classical-complement pathway c3/c5 convertase,complement factor I, complement factor D, alternative-complement pathwayc3/c5 convertase, cerevisin, hypodermin C, lysyl endopeptidase,endopeptidase 1a, gamma-reni, venombin AB, leucyl endopeptidase,tryptase, scutelarin, kexin, subtilisin, oryzin, endopeptidase K,thermomycolin, thermitase, endopeptidase SO, T-plasminogen activator,protein C, pancreatic endopeptidase E, pancreatic elastase II,IGA-specific serine endopeptidase, U-plasminogen, activator, venombin A,furin, myeloblastin, semenogelase, granzyme A or cytotoxic T-lymphocyteproteinase 1, granzyme B or cytotoxic T-lymphocyte proteinase 2,streptogrisin A, treptogrisin B, glutamyl endopeptidase II,oligopeptidase B, limulus clotting factor C, limulus clotting factor,limulus clotting enzyme, omptin, repressor lexa, bacterial leaderpeptidase I, and togavirin, flavirin. Cysteine proteinases includecathepsin B, papain, ficin, chymopapain, asclepain, clostripain,streptopain, actinide, cathepsin 1, cathepsin H, calpain, cathepsin T,glycyl, endopeptidase, cancer procoagulant, cathepsin S, picornain 3C,picornain 2A, caricain, ananain, stem bromelain, fruit bromelain,legumain, histolysain, and interleukin 1-beta converting enzyme.Aspartic proteinases include pepsin A, pepsin B, gastricsin, chymosin,cathepsin D, neopenthesin, renin, retropepsin, pro-opiomelanocortinconverting enzyme, aspergillopepsin I, aspergillopepsin II,penicillopepsin, rhizopuspepsin, endothiapepsin, mucoropepsin,candidapepsin, saccharopepsin, rhodotorulapepsin, physaropepsin,acrocylindropepsin, polyporopepsin, pycnoporopepsin, scytalidopepsin A,scytalidopepsin B, xanthomonapepsin, cathepsin E, barrierpepsin,bacterial leader peptidase I, pseudomonapepsin, and plasmepsin.Metalloproteinases include atrolysin A, microbial collagenase,leucolysin, interstitial collagenase, neprilysin, envelysin,IgA-specific metalloendopeptidase, procollagen N-endopeptidase, thimetoligopeptidase, neurolysin, stromelysin 1, meprin A, procollagenC-endopeptidase, peptidyl-lys metalloendopeptidase, astacin, stromelysin2, matrilysin gelatinase, aeromonolysin, pseudolysin, thermolysin,bacillolysin, aureolysin, coccolysin, mycolysin, beta-lyticmetalloendopeptidase, peptidyl-asp metalloendopeptidase, neutrophilcollagenase, gelatinase B, leishmanolysin, saccharolysin, autolysin,deuterolysin, serralysin, atrolysin B, atrolysin C, atroxase, atrolysinE, atrolysin F, adamalysin, horrilysin, ruberlysin, bothropasin,bothrolysin, ophiolysin, trimerelysin I, trimerelysin II, mucrolysin,pitrilysin, insulysin, O-syaloglycoprotein endopeptidase, russellysin,mitochondrial, intermediate, peptidase, dactylysin, nardilysin,magnolysin, meprin B, mitochondrial processing peptidase, macrophageelastase, choriolysin, and toxilysin. Proteinases of unknown mechanisminclude thermopsin and multicatalytic endopeptidase complex. Inembodiments, the second exogenous polypeptide comprises a fragment orvariant of any of the foregoing.

In embodiments, the second exogenous polypeptide comprises an IdeSpolypeptide. In some embodiments, the IdeS polypeptide comprises thesequence set out below as SEQ ID NO: 8 or a proteolytically activefragment of the sequence of SEQ ID NO: 8 (e.g., a fragment of at least100, 150, 200, 250, or 300 amino acids) or a sequence having at least70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to any of theforegoing. In some embodiments involving nucleic acids, the nucleic acidencodes an IdeS polypeptide having the sequence set out below as SEQ IDNO: 8, or a proteolytically active fragment thereof, or a sequencehaving at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to any of the foregoing.

IdeS polypeptide:

(SEQ ID NO: 8) DSFSANQEIRYSEVTPYHVTSVWTKGVTPPAKFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKEKIEAYLKKHPDKQKIMFGDQELLDVRKVINTKGDQTNSELFNYFRDKAFPGLSARRIGVMPDLVLDMFINGYYLNVYKTQTTDVNRTYQEKDRRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQ VLGLFTLSTGQDSWNQTN

In embodiments, the second exogenous polypeptide comprises a modifierdomain (e.g., a protease domain, e.g., an IdeS polypeptide) and amembrane anchor domain (e.g., a transmembrane domain, e.g., type I ortype II red blood cell transmembrane domain). In embodiments, themembrane anchor domain is C-terminal or N-terminal of the modifier(e.g., protease) domain. In embodiments, the transmembrane domaincomprises GPA or a transmembrane portion thereof. In embodiments, theGPA polypeptide has a sequence of:

(SEQ ID NO: 9) LSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ

or a transmembrane portion thereof, or a polypeptide having at least70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to any of theforegoing. In embodiments, the GPA polypeptide is C-terminal of themodifier (e.g., protease) domain.

In some embodiments, a linker is disposed between the IdeS polypeptideand the transmembrane polypeptide, e.g., a glycine-serine linker, e.g.,a linker comprising a sequence of GGSGGSGG (SEQ ID NO: 10) and/orGGGSGGGS (SEQ ID NO: 11).

In some embodiments, the exogenous polypeptide, e.g., the secondexogenous polypeptide, e.g., a protease, e.g., IdeS polypeptide,comprises a leader sequence, e.g., a GPA leader sequence, e.g.,MYGKIIFVLLLSEIVSISA (SEQ ID NO: 12).

In some embodiments, the exogenous polypeptide, e.g., the secondexogenous polypeptide further comprises a tag, e.g., an HA tag or a FLAGtag.

In some embodiments, the protease (e.g., immunoglobulin degradingenzyme, e.g., immunoglobulin-G degrading enzyme, e.g., IdeS) cleaves animmunoglobulin at a hinge region, a CH2 region, or between a hinge andCH2 region. In embodiments, the protease cleaves an immunoglobulin atone of the sequences below, e.g., between the two italicized glycines orthe italicized alanine and glycine in the sequences below.

Human IgG1 Hinge/CH2 Sequence (SEQ ID NO: 13) CPPCPAPELLGGPSVFHuman IgG2 Hinge/CH2 Sequence (SEQ ID NO: 14) CPPCPAPPVAGPSVFHuman IgG3 Hinge/CH2 Sequence (SEQ ID NO: 15) CPRCPAPELLGGPSVFHuman IgG4 Hinge/CH2 Sequence (SEQ ID NO: 16) AHHAQAPEFLGGPSVF

In embodiments, the protease (e.g., a bacterial protease) cleaves IgG,e.g., IdeS or IgA protease.

In embodiments, the protease (e.g., a papain family protease, e.g.,papain) cleaves an immunoglobulin between the Fc and Fab regions, e.g.,a histidine-threonine bond between positions 224 and 225 of the heavychain and/or a glutamic acid-leucine bond between positions 233 and 234of the heavy chain.

In embodiments, the protease or other modifier acts on a target listedin Table 4.

In embodiments, the second exogenous polypeptide comprises a catalyticmoiety described in WO2007030708, e.g., in pages 45-46 therein, whichapplication is herein incorporated by reference in its entirety.

The second exogenous polypeptide can comprise a moiety capable of actingon a target to induce a chemical change, thereby modulate its activity,e.g., a moiety capable of catalyzing a reaction within a target. Thesecond exogenous polypeptide can comprise a naturally occurring enzyme,an active (e.g., catalytically active) fragment thereof, or anengineered enzyme, e.g., a protein engineered to have an enzymaticactivity, such as a protein designed to contain a serine protease activemotif. A catalytic domain of a second exogenous polypeptide may comprisethe arrangement of amino acids that are effective to induce the desiredchemical change in the target. They may be N-terminal or C-terminaltruncated versions of natural enzymes, mutated versions, zymogens, orcomplete globular domains.

The second exogenous polypeptide can comprise an enzymatically activesite that alone is promiscuous, binding with a cleavage site itrecognizes on many different biomolecules, and may have relatively poorreaction kinetics. In embodiments, the first exogenous polypeptidesupplies or improves specificity by increasing the local concentrationof target near the second exogenous polypeptide.

The second exogenous polypeptide can, in embodiments, modify the targetso that it is recognized and acted upon by another enzyme (e.g., anenzyme that is already present in a subject). In an embodiment, thesecond exogenous polypeptide comprises a moiety that alters thestructure of the target so that its activity is inhibited orupregulated. Many naturally occurring enzymes activate other enzymes,and these can be exploited in accordance with the compositions andmethods described herein.

The second exogenous polypeptide can comprise a protease, a glycosidase,a lipase, or other hydrolases, an amidase (e.g.,N-acetylmuramoyl-L-alanine amidase, PGRP-L amidase), or other enzymaticactivity, including isomerases, transferases (including kinases),lyases, oxidoreductases, oxidases, aldolases, ketolases, glycosidases,transferases and the like. In embodiments, the second exogenouspolypeptide comprises human lysozyme, a functional portion of a humanlysozyme, a human PGRP-L, a functional portion of a human PGRP-L, aphospholipase A2, a functional portion of a phospholipase A2, or amatrix metalloproteinase (MMP) extracellular enzyme such as MMP-2(gelatinase A) or MMP-9 (gelatinase B).

In embodiments, the second exogenous polypeptide is a serine proteinase,e.g., of the chymotrypsin family which includes the mammalian enzymessuch as chymotrypsin, trypsin or elastase or kallikrein, or thesubstilisin family which includes the bacterial enzymes such assubtilisin. The general three-dimensional structure is different in thetwo families but they have the same active site geometry and catalysisproceeds via the same mechanism. The serine proteinases exhibitdifferent substrate specificities which are related to amino acidsubstitutions in the various enzyme subsites interacting with thesubstrate residues. Three residues which form the catalytic triad areimportant in the catalytic process: His-57, Asp-102 and Ser-195(chymotrypsinogen numbering).

In embodiments, the second exogenous polypeptide is a cysteineproteinase which includes the plant proteases such as papain, actinidinor bromelain, several mammalian lysosomal cathepsins, the cytosoliccalpains (calcium-activated), and several parasitic proteases (e.g.,Trypanosoma, Schistosoma). Papain is the archetype and the best studiedmember of the family. Like the serine proteinases, catalysis proceedsthrough the formation of a covalent intermediate and involves a cysteineand a histidine residue. The essential Cys-25 and His-159 (papainnumbering) play the same role as Ser-195 and His-57 respectively. Thenucleophile is a thiolate ion rather than a hydroxyl group. The thiolateion is stabilized through the formation of an ion pair with neighboringimidazolium group of His-159. The attacking nucleophile is thethiolate-imidazolium ion pair in both steps and then a water molecule isnot required.

In embodiments, the second exogenous polypeptide is an asparticproteinase, most of which belong to the pepsin family. The pepsin familyincludes digestive enzymes such as pepsin and chymosin as well aslysosomal cathepsins D, processing enzymes such as renin, and certainfungal proteases (penicillopepsin, rhizopuspepsin, endothiapepsin). Asecond family comprises viral proteinases such as the protease from theAIDS vims (HIV) also called retropepsin. In contrast to serine andcysteine proteinases, catalysis by aspartic proteinases does not involvea covalent intermediate, though a tetrahedral intermediate exists. Thenucleophilic attack is achieved by two simultaneous proton transfers:one from a water molecule to the dyad of the two carboxyl groups and asecond one from the dyad to the carbonyl oxygen of the substrate withthe concurrent CO—NH bond cleavage. This general acid-base catalysis,which may be called a “push-pull” mechanism leads to the formation of anon-covalent neutral tetrahedral intermediate.

In embodiments, the second exogenous polypeptide is a metalloproteinase,which can be found in bacteria, fungi as well as in higher organisms.They differ widely in their sequences and their structures but the greatmajority of enzymes contain a zinc (Zn) atom which is catalyticallyactive. In some cases, zinc may be replaced by another metal such ascobalt or nickel without loss of the activity. Bacterial thermolysin hasbeen well characterized and its crystallographic structure indicatesthat zinc is bound by two histidines and one glutamic acid. Many enzymescontain the sequence HEXXH, which provides two histidine ligands for thezinc whereas the third ligand is either a glutamic acid (thermolysin,neprilysin, alanyl aminopeptidase) or a histidine (astacin). Otherfamilies exhibit a distinct mode of binding of the Zn atom. Thecatalytic mechanism leads to the formation of a non-covalent tetrahedralintermediate after the attack of a zinc-bound water molecule on thecarbonyl group of the scissile bond. This intermediate is furtherdecomposed by transfer of the glutamic acid proton to the leaving group.

In embodiments, the second exogenous polypeptide comprises an isomerase(e.g., an isomerase that breaks and forms chemical bonds or catalyzes aconformational change). In embodiments, the isomerase is a racemase(e.g., amino acid racemase), epimerase, cis-trans isomerase,intramolecular oxidoreductase, intramolecular transferase, orintramolecular lyase.

In embodiments, the second exogenous protease comprises a chaperone. Forinstance, the chaperone can be a general chaperone (e.g., GRP78/BiP,GRP94, GRP170), a lectin chaperone (e.g., calnexin or calreticulin), anon-classical molecular chaperone (e.g., HSP47 or ERp29), a foldingchaperone (e.g., PDI, PPI, or ERp57), a bacterial or archaeal chaperone(e.g., Hsp60, GroEL/GroES complex, Hsp70, DnaK, Hsp90, HtpG, Hsp100, Clpfamily (e.g., ClpA and ClpX), Hsp104). In embodiments, the enucleatederythrocyte comprises a co-chaperone, e.g., immunophilin, Sti1, p50(Cdc37), or Ahal. In embodiments, the molecular chaperone is achaperonin.

Candidates for the second exogenous protein (which modifies a target)can be screened based on their activity. Depending on the specificactivity of each molecule being tested, an assay appropriate for thatmolecule can be used. For example, if the second exogenous protein is aprotease, the assay used to screen the protease can be an assay todetect cleavage products generated by the protease, e.g., achromatography or gel electrophoresis based assay.

In an example, the second exogenous polypeptide may have kinaseactivity. An assay for kinase activity could measure the amount ofphosphate that is covalently incorporated into the target of interest.For example, the phosphate incorporated into the target of interestcould be a radioisotope of phosphate that can be quantitated bymeasuring the emission of radiation using a scintillation counter.

Targets and Indications

In embodiments, the target is a target listed in Table 4.

In embodiments, the target is an immune checkpoint molecule selectedfrom PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAGS, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGF beta. Inembodiments, the target is an inhibitory ligand listed in Table 3, andthe first exogenous polypeptide optionally comprises a binding domainfrom a corresponding target receptor of Table 3. In some embodiments,the target is a target receptor of Table 3, and the first exogenouspolypeptide optionally comprises a binding domain from a correspondinginhibitory ligand of Table 3. In some embodiments, the second exogenouspolypeptide comprises a protease that cleaves an immune checkpointmolecule, e.g., trypsin. In embodiments, e.g., for treating cancer, a Tcell is activated or prevented from being inactivated, e.g., bycontacting its receptor (e.g., a receptor of Table 3) with a moleculethat blocks T cell inhibition.

In embodiments, the target is an antibody e.g., a human antibody.

Engineered erythroid cells described herein can also be used to treat asubject that has antibodies against a drug (e.g., see FIG. 7). Theerythroid cell can reduce levels of anti-drug antibodies in a subject,and can optionally further comprise a therapeutic protein that treatsthe disease. For instance, the erythroid cell comprises a firstexogenous polypeptide that binds a target, e.g., wherein the target isan anti-drug antibody. The erythroid cell can further comprise a secondexogenous polypeptide (e.g., IdeS) that inactivates, e.g., cleaves thetarget. The erythroid cell may optionally further comprise a thirdexogenous polypeptide, e.g., a therapeutic protein that treats the samedisease as the prior therapeutic to which the subject developedanti-drug antibodies, e.g., a therapeutic protein which is the same asor different from the prior therapeutic to which the subject developedanti-drug antibodies. In embodiments, the subject comprises anti-drugantibodies against an anti-CD20 antibody molecule, anti-VEGF-A antibodymolecule, anti-HER2 antibody molecule, an G-CSF analogue such asfilgrastim, anti EGFR antibody molecule (e.g., cetuximab), anerythropoietin, e.g., epoetin, or an interferon e.g., IFNβ1a or IFNβ1b.In such methods of treatment, the patient may be tested for the presenceof anti-drug antibodies, e.g., for the presence of neutralizinganti-drug antibodies, before, during and/or after administration of theengineered erythroid cells described herein.

Agent-Synergistic Configurations

When two or more agents (e.g., polypeptides) are agent-synergistic, theagents act on two or more different targets within a single pathway. Inan embodiment, the action of the two or more agents together is greaterthan the action of any of the individual agents. For example, the firstand second polypeptides are ligands for cellular receptors that signalto the same downstream target. For example, the first exogenouspolypeptide comprises a ligand for a first target cellular receptor, andthe second exogenous polypeptide comprises a ligand for a second targetcellular receptor, e.g., which first and second target cellularreceptors signal to the same downstream target. In embodiments, thefirst exogenous polypeptide acts on the first target and the secondexogenous polypeptide acts on the second target simultaneously, e.g.,there is some temporal overlap in binding of the first exogenouspolypeptide to the first target and binding of the second exogenouspolypeptide to the second target. In some embodiments the simultaneousaction generates a synergistic response of greater magnitude than wouldbe expected when either target is acted on alone or in isolation.

In an embodiment, the first and second polypeptides are ligands for afirst cellular receptor and a second cellular receptor that mediatesapoptosis. In an embodiment the agents comprise two or more TRAILreceptor ligands, e.g., wild-type or mutant TRAIL polypeptides, orantibody molecules that bind TRAIL receptors, and induce apoptosis in atarget cell, e.g., a cancer cell. In some embodiments, an enucleated RBCcomprising TRAIL receptor ligands is used to treat NSCLC. In someembodiments, a RBC comprising TRAIL receptor ligands further comprises atargeting moiety, e.g., a targeting moiety described herein. In anembodiment the first target and the second target interacts with thesame substrate, e.g., a substrate protein. In an embodiment the firsttarget and the second target interact with different substrates.

TRAIL (TNF-related apoptosis inducing ligand) is a member of the TNFfamily that induces apoptosis. TRAIL has at least two receptors, TRAILR1 and TRAIL R2. TRAIL receptor agonists, e.g., mutants of TRAIL thatbind one or more of the receptors, or antibody molecules that bind oneor both of TRAIL R1 or TRAIL R2 (see, e.g. Gasparian et al., Apoptosis2009 Jun. 14(6), Buchsbaum et al. Future Oncol 2007 Aug. 3(4)), havebeen developed as a clinical therapy for a wide range of cancers.Clinical trials of TRAIL receptor agonists have failed for, among otherreasons, the fact that many primary cancers are not sensitive tosignaling through a single receptor but rather require engagement ofboth receptors to induce cytotoxicity (Marconi et al., Cell Death andDisease (2013) 4, e863). In one embodiment the agents expressed on theengineered blood cell are single receptor-specific TRAIL agonists that,in combination, enable the cell to engage and agonize both TRAILreceptors simultaneously, thus leading to a synergistic induction ofapoptosis of a target cancer cell. Thus, in some embodiments, theenucleated red blood cell (e.g., reticulocyte) comprises on its surfacea first polypeptide that binds TRAIL R1 and a second polypeptide thatbinds TRAIL R2. In embodiments, each polypeptide has a Kd for TRAIL R1or TRAIL R2 that is 2, 3, 4, 5, 10, 20, 50, 100, 200, or 500-foldstronger than the Kd for the other receptor. While not wishing to bebound by theory, in some embodiments an enucleated red blood cellcomprising a TRAIL R1-specific ligand and a TRAIL R2-specific ligandpromote better heterodimerization of TRAIL R1 and TRAIL R2 than anenucleated red blood cell comprising a ligand that binds to TRAIL R1 andTRAIL R2 with about the same affinity.

In some embodiments, one, two, or more of the exogenous polypeptides aremembers of the TNF superfamily. In some embodiments, the exogenouspolypeptides bind to one or both of death receptors DR4 (TRAIL-R1) andDR5 (TRAIL-R2). In some embodiments, the exogenous polypeptides bind toone or more of TNFRSF10A/TRAILR1, TNFRSF10B/TRAILR2, TNFRSF10C/TRAILR3,TNFRSF10D/TRAILR4, or TNFRSF11B/OPG. In some embodiments, the exogenouspolypeptides activate one or more of MAPK8/JNK, caspase 8, and caspase3.

In some embodiments, a TRAIL polypeptide is a TRAIL agonist having asequence of any of SEQ ID NOS: 1-5 herein, or a sequence with at least70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.Sequence identity is measured, e.g., by BLAST (Basic Local AlignmentSearch Tool). SEQ ID Nos. 1-5 are further described in Mohr et al. BMCCancer (2015) 15:494), which is herein incorporated by reference in itsentirety.

Soluble TRAIL variant DR4-1 SEQ ID NO: 1MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACELKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRRRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG Soluble TRAIL variant DR4-2SEQ ID NO: 2 MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACELKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRRGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG Soluble TRAIL variant DR4-3SEQ ID NO: 3 MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACELKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRRRSNTLSSPNSKNEKALGIKINSWESSRRGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTDYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG Soluble TRAIL variant DR5-1SEQ ID NO: 4 MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACELKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMHHEASFFGAFLVG Soluble TRAIL variant DR5-2SEQ ID NO: 5 MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACELKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQERIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMHHEASFFGAFLVG

All combinations of the TRAIL receptor ligands are envisioned. In someembodiments, the first and second agents comprise SEQ ID NO: 1 and SEQID NO: 2; SEQ ID NO: 1 and SEQ ID NO: 3; SEQ ID NO: 1 and SEQ ID NO: 4;SEQ ID NO: 1 and SEQ ID NO: 5; SEQ ID NO: 2 and SEQ ID NO: 3; SEQ ID NO:2 and SEQ ID NO: 4; SEQ ID NO: 2 and SEQ ID NO: 5; SEQ ID NO: 3 and SEQID NO: 4; SEQ ID NO: 3 and SEQ ID NO: 5; or SEQ ID NO: 4 and SEQ ID NO:5, or a fragment or variant of any of the foregoing.

In some embodiments, the TRAIL receptor ligand comprises an antibodymolecule. In embodiments, the antibody molecule recognizes one or bothof TRAIL R1 and TRAIL R2. The antibody molecule may be, e.g.,Mapatumumab (human anti-DR4 mAb), Tigatuzumab (humanized anti-DR5 mAb),Lexatumumab (human anti-DR5 mAb), Conatumumab (human anti-DR5 mAb), orApomab (human anti-DR5 mAb). In some embodiments, the enucleated redblood cell (e.g., reticulocyte) comprises two or more (e.g., three,four, five, or more) different antibody molecules that bind a TRAILreceptor. In some embodiments, the enucleated red blood cell (e.g.,reticulocyte) comprises at least one antibody molecule that binds aTRAIL receptor and at least one TRAIL polypeptide.

In some embodiments, the agents are modulators of a multi-step pathwaythat act agent-synergistically by targeting upstream and downstreamsteps of the pathway, e.g., simultaneously.

Multiplicative Configurations

When two or more agents (e.g., polypeptides) are multiplicative, a firstagent acts on a first molecule that is part of a first pathway and asecond agent acts on a second molecule that is part of a second pathway,which pathways act in concert toward a desired response.

In some embodiments, the desired response is cell death, e.g., of acancer cell. Without wishing to be bound by theory, in cancer treatmentit may be beneficial to activate endogenous or exogenous anti-tumor Tcells that are anergic or otherwise non-functioning, e.g., due to thetumor or tumor microenvironment. In some embodiments, the agents triggermultiple T cell activation pathways to induce an anti-cancer immuneresponse. In some embodiments, the engineered erythroid cell promotes Tcell proliferation. In embodiments, one or more (e.g., 2, 3, 4, or 5 ormore) T cell activation ligands comprise a ligand of Table 2 or a T-cellactivating variant (e.g., fragment) thereof.

In embodiments, one or more (e.g., 2, 3, 4, or 5 or more) T cellactivation ligands comprise an antibody molecule that binds a targetreceptor of Table 2 or a T-cell activating variant (e.g., fragment)thereof. In some embodiments, the first and second polypeptides comprisedifferent T cell activation ligands, e.g. CD80, 41BB-ligand, CD86, orany combination thereof, to stimulate T cells and overcome anergy in animmuno-oncology setting. In some embodiments, the enucleated red bloodcell (e.g., reticulocyte) comprises 4-1BBL, OX40L, and CD40L, orfragments or variants thereof. In embodiments, these proteins signalthrough complementary activation pathways. In some embodiments theligands are activating cytokines, interferons, or TNF family members(e.g., of Table 1), e.g. IFNa, IL2, or IL6 or any combination thereof.In some embodiments the agents are combinations of the above classes ofmolecules. The agents can be derived from endogenous ligands or antibodymolecules to the target receptors.

TABLE 2 T cell activation Activating Ligand Target Receptor on T cellB7-H2 (e.g., Accession Number ICOS, CD28 (e.g., Accession NP_056074.1)Number NP_006130.1) B7-1 (e.g., Accession Number CD28 (e.g., AccessionNumber NP_005182.1) NP_006130.1) B7-2 (e.g., Accession Number CD28(e.g., Accession Number AAA86473) NP_006130.1) CD70 (e.g., AccessionNumber CD27 (e.g., Accession Number NP_001243.1) NP_001233.1) LIGHT(e.g., Accession Number HVEM (e.g., Accession Number NP_003798.2)AAQ89238.1) HVEM (e.g., Accession Number LIGHT (e.g., Accession NumberAAQ89238.1) NP_003798.2) CD40L (e.g., Accession Number CD40 (e.g.,Accession Number BAA06599.1) NP_001241.1) 4-1BBL (e.g., Accession Number4-1BB (e.g., Accession NP_003802.1) NP_001552.2) OX40L (e.g., AccessionNumber OX40 (e.g., Accession Number NP_003317.1) NP_003318.1) TL1A(e.g., Accession Number DR3 (e.g., Accession Number NP_005109.2)NP_683866.1) GITRL (e.g., Accession Number GITR (e.g., Accession NumberNP_005083.2) NP_004186.1) CD30L (e.g., Accession Number CD30 (e.g.,Accession Number NP_001235.1), NP_001234.3) TIM4 (e.g., Accession NumberTIM1 (e.g., Accession Number NP_612388.2) NP_036338.2) SLAM (e.g.,Accession Number SLAM (e.g., Accession Number AAK77968.1) AAK77968.1)CD48 (e.g., Accession Number CD2 (e.g., Accession Number CAG33293.1)NP_001315538.1) CD58 (e.g., Accession Number CD2 (e.g., Accession NumberCAG33220.1) NP_001315538.1) CD155 (e.g., Accession Number CD226 (e.g.,Accession Number NP_001129240.1) NP_006557.2) CD112 (e.g., AccessionNumber CD226 (e.g., Accession Number NP_001036189.1) NP_006557.2) CD137L(e.g., Accession Number CD137 (e.g., Accession NP_003802.1) NP_001552.2)

In some embodiments, an anti-IL6 or TNFa antibody molecule comprises asequence of either of SEQ ID NO: 6 or 7 herein, or a sequence with atleast 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

Anti-IL6 scFv SEQ ID NO: 6EVQLVESGGGLVQPGGSLRLSCAASGFNFNDYFMNWVRQAPGKGLEWVAQMRNKNYQYGTYYAESLEGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARESYYGFTSYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDIGISLSWYQQKPGKAPKLLIYNANNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHNSAPYTFGQGTKLEIKR Anti-TNFα scFvSEQ ID NO: 7 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK

As another example, the first and second polypeptides comprise a T cellactivating ligand and an agent which inhibits an immune inhibitorymolecule (e.g., an immune inhibitory receptor), e.g. CD80 and anti-PD1,in an immuno-oncology setting. In another embodiment, one agent is anactivating 4-1BBL, or fragment or variant thereof, and a second agent anantibody molecule that blocks PD1 signaling (e.g., an antibody moleculeto PD1 or PD-L1). Thus, in embodiments, a target T cell is bothactivated and prevented from being repressed. Examples of agents thatinhibit an immune inhibitory molecule include inhibitors of (e.g.,antibody molecules that bind) PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g.,CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1,CD160, 2B4 and TGF beta, or a functional variant (e.g., fragment)thereof. In some embodiments, the agent that inhibits an immuneinhibitory molecule is an inhibitor of an inhibitory ligand of Table 3,or an inhibitory fragment or variant thereof. In some embodiments, theagent that inhibits an immune inhibitory molecule is an antibodymolecule that binds a target receptor of Table 3, or a fragment orvariant thereof.

TABLE 3 T cell inhibition Inhibitory Ligand Target Receptor on T cellB7-1 CTLA4, B7H1 B7-2 CTLA4 B7DC PD1 B7H1 PD1, B7-1 HVEM CD160, BTLACOLLAGEN LAIR1 GALECTIN9 TIM3 CD48, TIM4 TIM4R CD48 2B4 CD155, CD112,CD113 TIGIT PDL1 PD1

In some embodiments, one of the agents for treating a cancer comprisesan activating cytokine, e.g., IL-2, IL-12, or another activatingcytokine of Table 1, or a fragment or variant thereof.

In some embodiments the objective is to activate or to inhibit T cells.To ensure that T cells are preferentially targeted over other immunecells that may also express either activating or inhibitory receptors asdescribed herein, one of the agents on the red blood cell (e.g.,reticulocyte) may comprise a targeting moiety, e.g., an antibodymolecule that binds the T cell receptor (TCR) or another T cell marker.Targeting moieties are described in more detail in the section entitled“Localization configurations” herein. In some embodiments, a specific Tcell subtype or clone may be enhanced (a T cell with anti-tumorspecificity) or inhibited. In some embodiments, one or more of theagents on the red blood cell (e.g., reticulocyte) is a peptide-MHCmolecule that will selectively bind to a T cell receptor in anantigen-specific manner.

In some embodiments a plurality of agents comprise multiple antigensderived from a complex target, e.g. a tumor cell, against which it isdesirable to mount a complex immune response with multiplespecificities.

In some embodiments, the first and second exogenous polypeptidescomprise, in some embodiments, an antigen and a costimulatory molecule,e.g., wherein the erythroid cell can act as an APC, e.g., for cancervaccination.

In some embodiments, an enucleated red blood cell (e.g., reticulocyte)comprising a first exogenous polypeptide and a second exogenouspolypeptide is administered to a subject having a first target and asecond target. In embodiments, the first exogenous polypeptide acts on(e.g., binds) the first target and the second exogenous polypeptide actson the second target. Optionally, the enucleated red blood cellcomprises a third exogenous polypeptide and the patient comprises athird target. In embodiments, the third exogenous polypeptide acts onthe third target.

In some embodiments an erythroid cell comprises a first exogenouspolypeptide which is an agonist or antagonist of a first target in afirst pathway, and further comprises a second exogenous polypeptidewhich is an agonist or antagonist of a second target in a secondpathway, wherein the first and second pathways act in concert toward adesired response. The first and second exogenous polypeptides can bothbe agonists; can both be antagonists; or one can be an agonist and theother can be an antagonist. In some embodiments, one or more of theexogenous polypeptides are immune checkpoint agonists or antagonists. Insome embodiments, the erythroid cell further comprises a targetingagent.

Independent Function Configurations

When two or more agents (e.g., polypeptides) have an independentfunction relationship, the agents have two distinct (e.g.,complementary) functions. For example, a first agent binds a firsttarget and the second agent binds a second target. The patient may lackthe first or second target. Optionally, the first and second agents arein different pathways.

In sepsis, tumor lysis syndrome, and other conditions marked by acytokine storm, the damage is driven by a diverse mix of inflammatorycytokines. Existing monotherapies against one cytokine are ofteninsufficient to treat these acute conditions. Furthermore it cansometimes be impossible to measure the driver of the cytokine storm intime to prevent clinical damage. In an embodiment, the first and secondpeptides are molecules (e.g., antibody molecules) that bind twodifferent cytokines. In some embodiments the agents bind and neutralizedifferent cytokines and thus the engineered red cell product providesmultifaceted protection from cytokine storm.

In embodiments the cytokines comprise interleukins, e.g., IL-1, IL02,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-25,IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, or IL-36.In some embodiments, the cytokine is a cytokine of Table 1 or a fragmentor variant thereof. In some embodiments, the first cytokine is TNFa andthe second is an interleukin, e.g., IL-6, or a fragment or variant ofany of the foregoing. In some embodiments, the agents compriseanti-TNFa, anti-IL-6, or anti-IFNg antibody molecules, or anycombination thereof, or a fragment or variant of any of the foregoing.

In some embodiments, an enucleated red blood cell (e.g., reticulocyte)comprising a first exogenous polypeptide and a second exogenouspolypeptide is administered to a subject having a first target but not asecond target, or wherein the patient is not known to have a firsttarget or second target. In embodiments, the first exogenous polypeptideacts on (e.g., binds) the first target and the second exogenouspolypeptide remains substantially unbound. Optionally, the enucleatedred blood cell comprises a third exogenous polypeptide and the patientlacks a third target, or is not known to have the third target. In someembodiments, the enucleated red blood cell comprises a plurality ofexogenous polypeptides, and the patient does not have, or is not knownto have, targets for one or a subset of the plurality of exogenouspolypeptides.

An example of an independent function configuration is shown in FIG. 4.The erythroid cell of FIG. 4 comprises a first exogenous polypeptide(white), a second exogenous polypeptide (hatching), and an optionalthird exogenous polypeptide (close hatching). The first exogenouspolypeptide can bind a first target, e.g., cytokine A, and the secondexogenous polypeptide can independently bind a second target, e.g.,cytokine B. This engineered erythroid cell trap and clear both cytokinesif both are present in the subject. If only one of the cytokines ispresent in the subject, the engineered erythroid cell can clear thatcytokine. In embodiments, one or more (e.g., two or all) of theexogenous polypeptides comprise antibody molecules, e.g., scFvs, andoptionally further comprise a transmembrane domain. In embodiments, thetargets comprise a plurality of cytokines, chemokines, or a combinationthereof.

Localization Configurations

When two or more agents (e.g., polypeptides) have a localizationrelationship, a first agent localizes the RBC to a site of action thatenhances the activity of the second or other agent or agents compared totheir activity when not localized to the site of action (e.g., bybinding of the first agent to its target, there is an increase in thelocal concentration of the second agent in the area of its target). Insome embodiments one agent serves to target the red blood cell (e.g.,reticulocyte) to a site of action and one or more agents have atherapeutic effect. In an embodiment, binding of the first agentincreases the activity of an entity, e.g., polypeptide, bound by thesecond agent. In an embodiment, the first agent binds to a substrate orproduct of the entity, e.g., polypeptide, bound by the second agent. Theagent that localizes the RBC may be, e.g., a ligand for a receptor on atarget cell, or an antibody that binds a cell surface molecule on atarget cell.

As shown in FIG. 9, the cell can comprise one or more targeting agents.The targeting agent can be an exogenous polypeptide. In embodiments, anerythroid cell comprises two targeting agents, which may increase thespecificity and/or affinity and/or avidity of the erythroid cell bindingto its target, compared to an otherwise similar erythroid cellcomprising only one of the targeting agents. The erythroid celloptionally further comprises an exogenous polypeptide with therapeuticactivity, e.g., anti-cancer activity. The exogenous polypeptide withtherapeutic activity can comprise an enzyme, capture reagent, agonist,or antagonist.

In embodiments, the targeting moiety comprises a receptor or a fragmentor variant thereof. In embodiments, the targeting moiety comprises anantibody molecule such as an scFv.

As another example, the targeting agent binds at or near a cancer cell,e.g., solid tumor cell, and the second agent (e.g., second polypeptide)has an anti-cancer function. In some embodiments the site of action istumor vasculature. In embodiments, the targeting agent binds a marker ofneovasculature, e.g. binds an integrin such as avB1, avB3, or avB5, ora4b1 integrins, e.g. a synthetic peptide knottin (Kim et al, JACS 2016,137(1)) or an endogenous or natural ligand, e.g. echistatin, RGD,EETI2.5F, or VCAM-1, or binds prostate-specific membrane antigen, whichis also found abundantly on neovasculature. In some embodiments, thetargeting agent binds a cancer cell marker such as CD269 (expressed,e.g., in multiple myeloma cells) or CD123 (expressed, e.g., in ALMcells), CD28 (expressed, e.g., in T cells; CD28 can be bound byCD80/CD86), NY-ESO-1 (expressed, e.g., in ovarian cancer).

The therapeutic agent may have, e.g., an anti-cancer effect, of whichthere are several strategies. For example the therapeutic agent may bean enzyme, e.g. asparaginase, methionine gamma lyase (MGL), serinedehyrodgenase, or fragment or variant thereof, that degrades metabolitesthat are selectively required by tumor cells to grow. The therapeuticagent may be an inhibitor of angiogenesis, e.g. an inhibitor ofangiopoitin or an inhibitor of VEGF or VEGFR to prevent further growthof blood vessels. The therapeutic agent may be an immunostimulatorymolecule to activate T cells, either a cytokine or a T cell activationligand (see, e.g., Table 1 and Table 2). The therapeutic agent may bindan immune effector cell, e.g. a T cell or an inflammatory macrophage andmay capture and bring the effector cell into proximity of the tumor. Thetherapeutic agent may be a direct mediator of cell killing, e.g. TRAILor FAS-L or other death ligands, or a toxin. In some embodiments, thetherapeutic agent comprises an agonist of a TRAIL receptor, e.g., anagonistic antibody molecule. In embodiments, the therapeutic agent is apro-apoptotic agent. In embodiments, the therapeutic agent comprises anadjuvant. For any of these therapeutic agents, the net result is a redcell therapeutic that localizes to a tumor site and thus concentratesits anti-tumor effect in a location that increases its efficacy.

In some embodiments, e.g., for treating a B cell cancer, the firstexogenous polypeptide comprises a surface-exposed anti-CD20 antibodymolecule that can target the cell to a cancer cell, and the secondexogenous polypeptide comprises a surface-exposed anti-CD40 antibodymolecule that can inhibit (e.g., kill) the cancer cell. The erythroidcell can further comprise an inhibitor of the anti-CD40 antibodymolecule, e.g., as illustrated in FIG. 8.

The first exogenous polypeptide can comprise a targeting agent and thesecond exogenous polypeptide can comprise an enzyme (e.g., FIG. 11). Forexample, in some embodiments, e.g., for treating a cancer, the erythroidcell comprises a first polypeptide comprising a targeting agent thatbinds a cancer cell and a second polypeptide that inhibits (e.g., kills,induces anergy in, inhibits growth of) the cancer cell. For instance,the targeting agent can comprise an anti-CD4 antibody which binds CD4 onthe surface of a T cell, e.g., a cancerous T cell. The secondpolypeptide can comprise an enzyme which can be surface-exposed orintracellular, e.g., intracellular and not membrane associated. Theenzyme may be IDO or a fragment or variant thereof, which depletestryptophan and can induce anergy in the cancerous T cell, or ADA or afragment or variant thereof. The enzyme may be a protease.

In embodiments, the target cell is an immune cell, e.g., a T cell, e.g.,a helper T cell, and/or a disease cell. The targeting agent may comprisean antibody molecule, e.g., an scFv.

The first exogenous polypeptide can comprise a targeting agent and thesecond exogenous polypeptide can comprise an agonist of a target (see,e.g., FIG. 12). In embodiments, the targeting agent comprises a receptoror fragment or variant thereof, an antibody molecule, a ligand orfragment or variant thereof, a cytokine or fragment or variant thereof.In embodiments, the second exogenous polypeptide comprises anattenuator, an activator, a cell-killing agent, or a cytotoxic molecule(e.g., a small molecule, protein, RNA e.g., antisense RNA, or TLRligand). In embodiments, the second exogenous polypeptide isintracellular, e.g., not membrane associated, and in some embodiments,the second exogenous polypeptide is surface-exposed.

The erythroid cell can comprise a targeting agent and a capture agent(e.g., FIG. 13). For example, the first exogenous polypeptide cancomprise a targeting agent that binds a plasma cell, e.g., an anti-BCMAantibody molecule. In embodiments, the second exogenous polypeptidebinds its target in a way that prevents the target from interacting withan endogenous receptor, e.g., binds the target at a moiety that overlapswith the receptor binding site. In embodiments, the targeting moietybinds a receptor at the site of disease. In embodiments, the targetingagent comprises a ligand or a cytokine or fragment or variant thereof,or an antibody molecule, e.g., an scFv. In embodiments, the captureagent comprises a receptor or fragment or variant thereof, or anantibody molecule, e.g., an scFv. In embodiments, the ligand is anunwanted cytokine or chemokine.

A targeting agent can direct an erythroid cell to a particular sub-typeof cell. The cell can further comprise a second exogenous polypeptidethat promotes a given activity or pathway in the target cell, e.g., canattenuate, activate, or induce cell death. For instance, FIG. 14 depictsan erythroid cell comprising a first exogenous polypeptide that can binda target cell. The erythroid cell can further comprise a secondexogenous polypeptide that inhibits (e.g., kills, or inhibits growth of)the cancer cell. For instance, the second exogenous polypeptide cancomprise a cell-killing agent, e.g., a pro-apoptotic agent, e.g., aTRAIL polypeptide that induces apoptosis in the cancer cell. Theerythroid cell may also comprise a targeting agent and an attenuator oractivator that is surface exposed or intracellular. For example, thecell can comprise a targeting agent and an enzyme such as IDO or ADA ora fragment or variant thereof.

Proximity-Based Configurations

When two or more agents (e.g., polypeptides) have a proximity-basedrelationship, the two agents function more strongly, e.g., exert a morepronounced effect, when they are in proximity to each other than whenthey are physically separate. In embodiments, the two agents are inproximity when they are directly binding to each other, when they arepart of a complex (e.g., linked by a third agent), when they are presenton the same cell membrane, or when they are present on the samesubsection of a cell membrane (e.g., within a lipid raft, outside alipid raft, or bound directly or indirectly to an intracellularstructure such as a cytoskeleton component). In some embodiments, firstpolypeptide binds a first target molecule and the second polypeptidebinds a second target molecule, and this binding causes the first targetmolecule and the second target molecule to move into closer proximitywith each other, e.g., to bind each other. In some embodiments, thefirst and second target molecules are cell surface receptors on a targetcells.

An example of a proximity-based configuration is shown in FIG. 4. Theerythroid cell of FIG. 4 comprises an optional first exogenouspolypeptide (white), a second exogenous polypeptide (light gray), and athird exogenous polypeptide (dark gray). The second and third exogenouspolypeptides bind to different epitopes within the same polypeptidechain of a target, e.g., cytokine B. The second and third exogenouspolypeptides, which are mounted on the erythrocyte, bind to the targetwith higher avidity than if the second and third exogenous polypeptideswere free polypeptides. As examples, two or more exogenous polypeptidescould bind different sites on the same target, wherein the target is acytokine, an enzyme, or an antibody.

Scaffold Configurations

When two or more agents (e.g., polypeptides) have a scaffoldrelationship, the agents bring two or more targets together, to increasethe likelihood of the targets interacting with each other. In anembodiment the first and second agent are associated with each other(forming a scaffold) at the surface of the RBC, e.g., two complexedpolypeptides. In an embodiment, the red blood cell (e.g., reticulocyte)comprises a bispecific antibody molecule, e.g., an antibody moleculethat recognizes one or more (e.g., 2) proteins described herein, e.g.,in any of Table 1, Table 2, and Table 3.

The targets may comprise, e.g., proteins, cells, small molecules, or anycombination thereof. In an embodiment, the first and second targets areproteins. In an embodiment, the first and second targets are cells.

As another example, a RBC brings an immune effector cell (e.g., T cell)and a cancer cell in close proximity with one another to facilitate thekilling of the cancer cell by the immune effector cell. Thus, in someembodiments, the first polypeptide binds a cell surface marker of acancer cell and the second polypeptide binds a cell surface marker of animmune effector cell. The first and second polypeptides may comprise,e.g., antibody molecules. In some embodiments, the cancer cell marker isselected from CD19 (expressed, e.g., in B cell acute leukemia), EpCAM(expressed, e.g., in CTCs), CD20 (expressed, e.g., in B cell acuteleukemia), CD45 (expressed, e.g., in CTCs), EGFR, HER2 (expressed, e.g.,in breast cancer cells). In some embodiments, the immune cell marker isCD3.

In some embodiments, the RBC brings an immune effector cell intoproximity with another immune cell, e.g., to promote antigenpresentation (e.g., when one cell is an antigen presenting cell and theother cell is a T cell), e.g., for a cancer vaccine.

In some embodiments, a RBC expresses an exogenous fusion polypeptidecomprising a first antibody molecule domain and a second antibodymolecule domain, wherein the exogenous polypeptide functions as abispecific antibody, e.g., wherein the first antibody molecule domainbinds a first target on a first cell and the second antibody moleculedomain binds a second target on a second cell, e.g., a different celltype.

Multimer Configurations

When two or more agents (e.g., polypeptides) have a multimerconfiguration, the agents combine with each other, e.g., bind eachother, to form a complex that has a function or activity on a target. Inan embodiment, the agents are subunits of a cell surface complex, e.g.,MHCI, and a function is to bind a peptide. In an embodiment, the agentsare subunits of MHCII, and a function is to bind a peptide. In anembodiment, the agents are subunits of a cell surface molecule, e.g.,MHCI and a peptide, e.g., a peptide loaded on the MHCI molecule, and afunction is to present the peptide. In an embodiment, the agents aresubunits of a MHCII and a peptide, e.g., a peptide loaded on the MHCIImolecule, and a function is to present the peptide. In one embodiment,the complex is a functional MHC I, the agents are MHC I (alpha chain1-3) and beta-2 microglobulin, or fragments or variants thereof. In oneembodiment the complex is MHC II and the agents are MHC II alpha chainand MHC II beta chain, or fragments or variants thereof. In someembodiments, the MHC molecule comprises human MHC class I or II, e.g.,MHC II alpha subunit and MHC II beta subunit or a fusion moleculecomprising both subunits or antigen-presenting fragments thereof. A RBCwith these two polypeptides is used, in some embodiments, for immuneinduction or antigen presentation. In some embodiments, the RBCcomprises a single protein that is a fusion between an MHC molecule andan antigen, e.g., a single-chain peptide-MHC construct. In someembodiments, a non-membrane tethered component of the complex, e.g. thepeptide, or the beta-2 microglobulin, is assembled with another agentwithin the cell prior to trafficking to the surface, is secreted by thecell then captured on the surface by the membrane-tethered component ofthe multimer, or is added in a purified form to an engineered red bloodcell.

The antigen is, in some embodiments, a cancer antigen, e.g., for acancer vaccine. In some embodiments, the antigen is about 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or 35 amino acids in length.

In some embodiments the complex comprises multiple subdomains derivedfrom different polypeptide chains, all of which must be expressed inorder for the complex to be active.

Compensatory Configurations

When two or more agents (e.g., polypeptides) have a compensatoryrelationship, a first agent reduces an undesirable characteristic of asecond agent. For example, in some embodiments, the second agent has agiven level of immunogenicity, and the first agent reduces theimmunogenicity, e.g., by negatively signaling immune cells (see Table3), or by shielding an antigenic epitope of the second agent. In someembodiments, the second agent has a given half-life, and the first agentincreases the half-life of the second agent. For example, the firstagent can comprise a chaperone or fragment or variant thereof.

An enucleated erythroid cell can co-express a therapeutic protein andits inhibitor (e.g., FIG. 8). The inhibitor can be released (e.g., ceasebinding the therapeutic but remain on the surface of the cell) in at thedesired location in the body, to activate the therapeutic protein.

For instance, in some embodiments, the erythroid cell comprises a firstexogenous polypeptide with therapeutic activity (e.g., an anti-CD40antibody molecule), a second exogenous polypeptide (e.g., CD40 or afragment or variant thereof) that inhibits the first exogenouspolypeptide. The second polypeptide (e.g., CD40) may inhibit activity ofthe first exogenous polypeptide (e.g., anti-CD40) until the erythroidcell is at a desired location, e.g., a cancer cell, e.g., limitingoff-target effects. The second exogenous polypeptide (e.g., CD40 or avariant thereof) may comprise a variant of the target (e.g., endogenousCD40) that the first exogenous polypeptide (e.g., anti-CD40) binds. Forinstance, the variant can be a weakly-binding variant that is competedaway in the presence of the target. In embodiments, the Kd of the firstexogenous polypeptide for the second exogenous polypeptide is at least2, 3, 5, 10, 20, 50, or 100-fold greater than the Kd of the firstexogenous polypeptide for its target. The erythroid cell optionallycomprises a third exogenous polypeptide that comprises a targetingagent, e.g., an anti-CD20 antibody molecule.

In some embodiments, the enucleated erythroid cell comprises a prodrug(e.g., pro-insulin) that becomes a drug (e.g., insulin) at a desiredsite in a subject.

Enucleated Red Blood Cells Comprising Three or More Agents (e.g.,Polypeptides)

In embodiments, a red blood cell (e.g., reticulocyte) described hereincomprises three or more, e.g., at least 4, 5, 10, 20, 50, 100, 200, 500,or 1000 agents. In embodiments, a population of red blood cellsdescribed herein comprises three or more, e.g., at least 4, 5, 10, 20,50, 100, 200, 500, 1000, 2000, or 5000 agents, e.g., wherein differentRBCs in the population comprise different agents or wherein differentRBCs in the population comprise different pluralities of agents. Inembodiments, two or more (e.g., all) of the agents in the RBC orpopulation or RBCs have agent-additive, agent-synergistic,multiplicative, independent function, localization-based,proximity-dependent, scaffold-based, multimer-based, or compensatoryactivity.

In embodiments, the RBC is produced by contacting a RBC progenitor cellwith a plurality of mRNAs encoding the agents.

Eukaryotic Display Screening.

In an embodiment, a combinatorial, high-diversity pool of cells isproduced, e.g., for use in an in vitro or in vivo binding assay. Acombinatorial, high-diversity nucleic acid library encoding cell-surfaceproteins can be created. Such a library could, e.g., consist of entirelyvariable sequences, or comprise a fixed sequence fused to a highlyvariable, combinatorial sequence. These can be introduced into red bloodcell progenitors as a mixture or individually, using methods such aselectroporation, transfection or viral transduction. In one embodiment,the cells are subsequently grown in differentiation media until thedesired level of maturity. In one embodiment, the cells are used for ahighly multiplexed in-vitro assay. Cells are incubated with a biologicalsample in a microtiter plate. Wells are washed using a cell-compatiblebuffer, with a desired level of stringency. The remaining cells areisolated and analyzed for the enrichment of specific sequences. In oneembodiment, the analysis is performed at the protein level, e.g., usingmass spectrometry, to identify the amino acid motifs that are enrichedin the bound population. In an embodiment, the analysis is performed atthe nucleic acid level (RNA or DNA) to identify the nucleic acidsequences identifying the corresponding amino-acid motif enriched in thebound population. In an embodiment, the high-diversity cell populationis injected into an animal model (either healthy or diseased). In oneembodiment the cells are fluorescently labeled to visualize their invivo distribution or localization. Various tissues of the animal couldthen be collected and analyzed for the relative enrichment of specificamino-acid motifs or nucleic acid sequences identifying thecorresponding amino-acid motif.

Expression Optimization.

A large number of variants can be simultaneously transfected intoindividual cells to assess their relative transcription or translationability. In embodiments, a library of protein coding sequences aredesigned and synthesized with a diversity of 5′ untranslated regions, 3′untranslated regions, codon representations, amino acid changes, andother sequence differences. This library would be introduced into redblood cell progenitors as a mixture or individually, using methods suchas electroporation, transfection or viral transduction. In oneembodiment, the cells are subsequently grown in differentiation mediauntil the desired level of maturity.

Physical Characteristics of Enucleated Red Blood Cells

In some embodiments, the RBCs (e.g., reticulocytes) described hereinhave one or more (e.g., 2, 3, 4, or more) physical characteristicsdescribed herein, e.g., osmotic fragility, cell size, hemoglobinconcentration, or phosphatidylserine content. While not wishing to bebound by theory, in some embodiments an enucleated RBC that expresses anexogenous protein has physical characteristics that resemble awild-type, untreated RBC. In contrast, a hypotonically loaded RBCsometimes displays aberrant physical characteristics such as increasedosmotic fragility, altered cell size, reduced hemoglobin concentration,or increased phosphatidylserine levels on the outer leaflet of the cellmembrane.

In some embodiments, the enucleated RBC comprises an exogenous proteinthat was encoded by an exogenous nucleic acid that was not retained bythe cell, has not been purified, or has not existed fully outside anRBC. In some embodiments, the RBC is in a composition that lacks astabilizer.

Osmotic Fragility

In some embodiments, the enucleated red blood cell exhibitssubstantially the same osmotic membrane fragility as an isolated,uncultured erythroid cell that does not comprise an exogenouspolypeptide. In some embodiments, the population of enucleated red bloodcells has an osmotic fragility of less than 50% cell lysis at 0.3%,0.35%, 0.4%, 0.45%, or 0.5% NaCl. Osmotic fragility can be assayed usingthe method of Example 59 of WO2015/073587.

Cell Size

In some embodiments, the enucleated RBC has approximately the diameteror volume as a wild-type, untreated RBC.

In some embodiments, the population of RBC has an average diameter ofabout 4, 5, 6, 7, or 8 microns, and optionally the standard deviation ofthe population is less than 1, 2, or 3 microns. In some embodiments, theone or more RBC has a diameter of about 4-8, 5-7, or about 6 microns. Insome embodiments, the diameter of the RBC is less than about 1 micron,larger than about 20 microns, between about 1 micron and about 20microns, between about 2 microns and about 20 microns, between about 3microns and about 20 microns, between about 4 microns and about 20microns, between about 5 microns and about 20 microns, between about 6microns and about 20 microns, between about 5 microns and about 15microns or between about 10 microns and about 30 microns. Cell diameteris measured, in some embodiments, using an Advia 120 hematology system.

In some embodiment the volume of the mean corpuscular volume of the RBCsis greater than 10 fL, 20 fL, 30 fL, 40 fL, 50 fL, 60 fL, 70 fL, 80 fL,90 fL, 100 fL, 110 fL, 120 fL, 130 fL, 140 fL, 150 fL, or greater than150 fL. In one embodiment the mean corpuscular volume of the RBCs isless than 30 fL, 40 fL, 50 fL, 60 fL, 70 fL, 80 fL, 90 fL, 100 fL, 110fL, 120 fL, 130 fL, 140 fL, 150 fL, 160 fL, 170 fL, 180 fL, 190 fL, 200fL, or less than 200 fL. In one embodiment the mean corpuscular volumeof the RBCs is between 80-100, 100-200, 200-300, 300-400, or 400-500femtoliters (fL). In some embodiments, a population of RBCs has a meancorpuscular volume set out in this paragraph and the standard deviationof the population is less than 50, 40, 30, 20, 10, 5, or 2 fL. The meancorpuscular volume is measured, in some embodiments, using ahematological analysis instrument, e.g., a Coulter counter.

Hemoglobin Concentration

In some embodiments, the enucleated RBC has a hemoglobin content similarto a wild-type, untreated RBC. In some embodiments, the RBCs comprisegreater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or greater than 10%fetal hemoglobin. In some embodiments, the RBCs comprise at least about20, 22, 24, 26, 28, or 30 pg, and optionally up to about 30 pg, of totalhemoglobin. Hemoglobin levels are determined, in some embodiments, usingthe Drabkin's reagent method of Example 33 of WO2015/073587.

Phosphatidylserine Content

In some embodiments, the enucleated RBC has approximately the samephosphatidylserine content on the outer leaflet of its cell membrane asa wild-type, untreated RBC. Phosphatidylserine is predominantly on theinner leaflet of the cell membrane of wild-type, untreated RBCs, andhypotonic loading can cause the phosphatidylserine to distribute to theouter leaflet where it can trigger an immune response. In someembodiments, the population of RBC comprises less than about 30, 25, 20,15, 10, 9, 8, 6, 5, 4, 3, 2, or 1% of cells that are positive forAnnexin V staining. Phosphatidylserine exposure is assessed, in someembodiments, by staining for Annexin-V-FITC, which binds preferentiallyto PS, and measuring FITC fluorescence by flow cytometry, e.g., usingthe method of Example 54 of WO2015/073587.

Other Characteristics

In some embodiments, the population of RBC comprises at least about 50%,60%, 70%, 80%, 90%, or 95% (and optionally up to 90 or 100%) of cellsthat are positive for GPA. The presence of GPA is detected, in someembodiments, using FACS.

In some embodiments, the RBCs have a half-life of at least 30, 45, or 90days in a subject.

In some embodiments, a population of cells comprising RBCs comprisesless than about 10, 5, 4, 3, 2, or 1% echinocytes.

In some embodiments, an RBC is enucleated, e.g., a population of cellscomprising RBCs used as a therapeutic preparation described herein isgreater than 50%, 60%, 70%, 80%, 90% enucleated. In some embodiments, acell, e.g., an RBC, contains a nucleus that is non-functional, e.g., hasbeen inactivated.

Methods of Manufacturing Enucleated Red Blood Cells

Methods of manufacturing enucleated red blood cells (e.g.,reticulocytes) comprising (e.g., expressing) exogenous agent (e.g.,polypeptides) are described, e.g., in WO2015/073587 and WO2015/153102,each of which is incorporated by reference in its entirety.

In some embodiments, hematopoietic progenitor cells, e.g., CD34+hematopoietic progenitor cells, are contacted with a nucleic acid ornucleic acids encoding one or more exogenous polypeptides, and the cellsare allowed to expand and differentiate in culture.

In some embodiments, the two or more polypeptides are encoded in asingle nucleic acid, e.g. a single vector. In embodiments, the singlevector has a separate promoter for each gene, has two proteins that areinitially transcribed into a single polypeptide having a proteasecleavage site in the middle, so that subsequent proteolytic processingyields two proteins, or any other suitable configuration. In someembodiments, the two or more polypeptides are encoded in two or morenucleic acids, e.g., each vector encodes one of the polypeptides.

The nucleic acid may be, e.g., DNA or RNA. A number of viruses may beused as gene transfer vehicles including retroviruses, Moloney murineleukemia virus (MMLV), adenovirus, adeno-associated virus (AAV), herpessimplex virus (HSV), lentiviruses such as human immunodeficiency virus 1(HIV 1), and spumaviruses such as foamy viruses, for example.

In some embodiments, the cells are produced using sortagging, e.g., asdescribed in WO2014/183071 or WO2014/183066, each of which isincorporated by reference in its entirety.

In some embodiments, the RBCs are expanded at least 1000, 2000, 5000,10,000, 20,000, 50,000, or 100,000 fold (and optionally up to 100,000,200,000, or 500,000 fold). Number of cells is measured, in someembodiments, using an automated cell counter.

In some embodiments, the population of RBC comprises at least 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 98% (and optionallyup to about 80, 90, or 100%) enucleated RBC. In some embodiments, thepopulation of RBC contains less than 1% live enucleated cells, e.g.,contains no detectable live enucleated cells. Enucleation is measured,in some embodiments, by FACS using a nuclear stain. In some embodiments,at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% (and optionallyup to about 70, 80, 90, or 100%) of RBC in the population comprise oneor more (e.g., 2, 3, 4 or more) of the exogenous polypeptides.Expression of the polypeptides is measured, in some embodiments, by FACSusing labeled antibodies against the polypeptides. In some embodiments,at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% (and optionallyup to about 70, 80, 90, or 100%) of RBC in the population are enucleatedand comprise one or more (e.g., 2, 3, 4, or more) of the exogenouspolypeptides. In some embodiments, the population of RBC comprises about1×10⁹-2×10⁹, 2×10⁹-5×10⁹, 5×10⁹-1×10¹⁰, 1×10¹⁰-2×10¹⁰, 2×10¹⁰-5×10¹⁰,5×10¹⁰-1×10¹¹, 1×10¹¹-2×10¹¹, 2×10¹¹-5×10¹¹, 5×10¹¹-1×10¹²,1×10¹²-2×10¹², 2×10¹²-5×10¹², or 5×10¹²-1×10¹³ cells.

Physically Proximal, Synergistic Agents

In some aspects, the present disclosure provides a compositioncomprising a first agent and a second agent in physical proximity toeach other. In some embodiments, agents act synergistically when theyare in physical proximity to each other but not when they are separate.In some embodiments, the first and second agent are covalently linked,e.g., are part of a fusion protein or are chemically conjugatedtogether. In some embodiments, the first and second agent arenon-covalently linked, e.g., are bound directly to each other or to ascaffold. In some embodiments, the first and second agents are part of(e.g., linked to) a nanoparticle (e.g., 1-100, 100-2,500, or2,500-10,000 nm in diameter) liposome, vesicle, bead, polymer, implant,or polypeptide complex.

In some embodiments, the composition comprises at least 3, 4, 5, 6, 7,8, 9, or 10 different agents that are in physical proximity to eachother (e.g., covalently or noncovalently linked).

In some embodiments, the composition comprises one or more (e.g., 2, 3,4, 5, or more) agents described herein, e.g., exogenous polypeptidesdescribed herein, e.g., polypeptides of any of Table 1, Table 2, orTable 3, or a fragment or variant thereof, or an antibody moleculethereto. In some embodiments, one or more (e.g., 2, 3, or more) of theexogenous polypeptides comprise cytokines, interleukins, cytokinereceptors, Fc-binding molecules, T-cell activating ligands, T cellreceptors, immune inhibitory molecules, costimulatory molecule, MHCmolecules, APC-binding molecule, toxin, targeting agent, anti-canceragent, cancer cell marker, agent that binds a cancer cell marker, orTRAIL receptor ligands.

In some embodiments, one or more (e.g., 2, 3, or more) of the exogenouspolypeptides comprise TRAIL receptor ligands, e.g., a sequence of any ofSEQ ID NOS: 1-5 herein, or a sequence with at least 70%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identity thereto, or an antibody moleculethat binds a TRAIL receptor. In some embodiments, the first agent bindsto a first TRAIL receptor, e.g., TRAIL-RI, and the second agent binds toa second TRAIL receptor, e.g., TRAIL-RII. In embodiments, the two TRAILreceptor ligands in proximity provide a synergistic degree of apoptosisin a target cell, compared to either TRAIL receptor ligand alone.Example 1 herein demonstrates a synergistic activity when cancer cellsare treated with a composition comprising two TRAIL receptor ligands inclose proximity (e.g., on the surface of an enucleated red blood cell).

Engineered Red Blood Cells Comprising One or More Agents

In some aspects, the present disclosure provides an engineered red bloodcell (e.g., reticulocyte) comprising an exogenous agent. Morespecifically, in some aspects, the present disclosure provides anenucleated red blood cell (e.g., reticulocyte) comprising an exogenouspolypeptide. The red blood cell optionally further comprises a second,different, exogenous polypeptide.

In some embodiments, the exogenous polypeptide (e.g., an exogenouspolypeptide comprised by a red blood cell that optionally furthercomprises a second exogenous polypeptide) is an exogenous polypeptidedescribed herein. In embodiments, the polypeptide is selected from anyof Table 1, Table 2, or Table 3, or a fragment or variant thereof, or anantibody molecule thereto.

In some embodiments, the exogenous polypeptide (e.g., an exogenouspolypeptide comprised by a red blood cell that optionally furthercomprises a second exogenous polypeptide) comprises a stimulatoryligand, e.g., CD80, CD86, 41BBL, or any combination thereof, e.g., forthe treatment of a cancer. In some embodiments, the exogenouspolypeptide comprises a cancer cell antigen such as CD269, e.g., for thetreatment of a cancer such as multiple myeloma.

In some embodiments, the exogenous polypeptide (e.g., an exogenouspolypeptide comprised by a red blood cell that optionally furthercomprises a second exogenous polypeptide) inhibits an immune checkpointmolecule. In embodiments, the exogenous polypeptide is situated at thesurface of the engineered red blood cell (e.g., comprises atransmembrane portion and a surface-exposed portion) and binds an immunecheckpoint molecule. In embodiments, the immune checkpoint molecule isPD-1 or PD-L1. In embodiments, the immune checkpoint molecule is PD1,PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5),LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGF beta. In someembodiments, the exogenous polypeptide (e.g., an exogenous polypeptidecomprised by a red blood cell that optionally further comprises a secondexogenous polypeptide) inhibits an immune checkpoint molecule. In oneembodiment, the inhibitor of the immune checkpoint molecule is aninhibitory antibody molecule (e.g., an antibody such as a monospecificantibody, monoclonal antibody, or a single chain antibody). The antibodymolecule may be, e.g., humanized or fully human. In other embodiments,the inhibitor of the immune checkpoint molecule is a fusion protein,e.g., an Fc-receptor fusion protein. In some embodiments, the inhibitorof the immune checkpoint molecule is an agent, such as an antibodymolecule, that interacts with an immune checkpoint protein. In someembodiments, the inhibitor of the immune checkpoint molecule is anagent, such as an antibody molecule, that interacts with the ligand ofan immune checkpoint receptor. In one embodiment, the inhibitor of theimmune checkpoint molecule is an inhibitor (e.g., an inhibitory antibodyor small molecule inhibitor) of CTLA-4 (e.g., an anti-CTLA4 antibodysuch as ipilimumab/Yervoy or tremelimumab). In one embodiment, theinhibitor of the immune checkpoint molecule is an inhibitor (e.g., aninhibitory antibody or small molecule inhibitor) of PD-1 (e.g.,nivolumab/Opdivo®; pembrolizumab/Keytruda®; pidilizumab/CT-011). In oneembodiment, the inhibitor of the immune checkpoint molecule is aninhibitor (e.g., an inhibitory antibody or small molecule inhibitor) ofPD-L1 (e.g., MPDL3280A/RG7446; MEDI4736; MSB0010718C; BMS 936559). Inone embodiment, the inhibitor of the immune checkpoint molecule is aninhibitor (e.g., an inhibitory antibody or Fc fusion or small moleculeinhibitor) of PDL2 (e.g., a PDL2/Ig fusion protein such as AMP 224). Inone embodiment, the inhibitor of the immune checkpoint molecule is aninhibitor (e.g., an inhibitory antibody or small molecule inhibitor) ofB7-H3 (e.g., MGA271), B7-H4, BTLA, HVEM, TIM3, GALS, LAGS, VISTA, KIR,2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands, or acombination thereof.

Inhibitors of immune checkpoint molecules can be broken down into atleast 4 major categories: i) agents such as antibody molecules thatblock an inhibitory pathway directly on T cells or natural killer (NK)cells (e.g., PD-1 targeting antibodies such as nivolumab andpembrolizumab, antibodies targeting TIM-3, and antibodies targetingLAG-3, 2B4, CD160, A2aR, BTLA, CGEN-15049, or KIR), ii) agents such asantibodies that activate stimulatory pathways directly on T cells or NKcells (e.g., antibodies targeting OX40, GITR, or 4-1BB), iii) agentssuch as antibody molecules that block a suppressive pathway on immunecells or rely on antibody-dependent cellular cytotoxicity to depletesuppressive populations of immune cells (e.g., CTLA-4 targetingantibodies such as ipilimumab, antibodies targeting VISTA, andantibodies targeting PD-L2, Gr1, or Ly6G), and iv) agents such asantibody molecules that block a suppressive pathway directly on cancercells or that rely on antibody-dependent cellular cytotoxicity toenhance cytotoxicity to cancer cells (e.g., rituximab, antibodiestargeting PD-L1, and antibodies targeting B7-H3, B7-H4, Gal-9, or MUC1).Such agents described herein can be designed and produced, e.g., byconventional methods known in the art (e.g., Templeton, Gene and CellTherapy, 2015; Green and Sambrook, Molecular Cloning, 2012).

Vehicles for Polypeptides Described Herein

While in many embodiments herein, the one or more (e.g., two or more)exogenous polypeptides are situated on or in a red blood cell, it isunderstood that any exogenous polypeptide or combination of exogenouspolypeptides described herein can also be situated on or in anothervehicle. The vehicle can comprise, e.g., a cell, an erythroid cell, acorpuscle, a nanoparticle, a micelle, a liposome, or an exosome. Forinstance, in some aspects, the present disclosure provides a vehicle(e.g., a cell, an erythroid cell, a corpuscle, a nanoparticle, amicelle, a liposome, or an exosome) comprising, e.g., on its surface,one or more agents described herein. In some embodiments, the one ormore agent comprises a polypeptide that binds PD-1 (e.g., an antibodymolecule that binds PD-1 or an agonist of PD-1 such as PD-L1), apolypeptide that binds PD-L1 (e.g., an antibody molecule that bindsPD-L1), a polypeptide that binds CD20 (e.g., an antibody molecule thatbinds CD20), or a polypeptide that binds a TRAIL receptor (e.g., anagonist of a TRAIL receptor). In some embodiments, the one or moreagents comprise an agent selected a polypeptide of any of Table 1, Table2, or Table 3, or a fragment or variant thereof, or an agonist orantagonist thereof, or an antibody molecule thereto. In someembodiments, the vehicle comprises two or more agents described herein,e.g., any pair of agents described herein.

In some embodiments, the vehicle comprises an erythroid cell. Inembodiments, the erythroid cell is a nucleated red blood cell, red bloodcell precursor, or enucleated red blood cell. In embodiments, theerythroid cell is a cord blood stem cell, a CD34+ cell, a hematopoieticstem cell (HSC), a spleen colony forming (CFU-S) cell, a common myeloidprogenitor (CMP) cell, a blastocyte colony-forming cell, a burst formingunit-erythroid (BFU-E), a megakaryocyte-erythroid progenitor (MEP) cell,an erythroid colony-forming unit (CFU-E), a reticulocyte, anerythrocyte, an induced pluripotent stem cell (iPSC), a mesenchymal stemcell (MSC), a polychromatic normoblast, an orthochromatic normoblast, ora combination thereof. In some embodiments, the erythroid cells areimmortal or immortalized cells.

Cells Encapsulated in a Membrane

In some embodiments, enucleated erythroid cells or other vehiclesdescribed herein are encapsulated in a membrane, e.g., semi-permeablemembrane. In embodiments, the membrane comprises a polysaccharide, e.g.,an anionic polysaccharide alginate. In embodiments, the semipermeablemembrane does not allow cells to pass through, but allows passage ofsmall molecules or macromolecules, e.g., metabolites, proteins, or DNA.In embodiments, the membrane is one described in Lienert et al.,“Synthetic biology in mammalian cells: next generation research toolsand therapeutics” Nature Reviews Molecular Cell Biology 15, 95-107(2014), incorporated herein by reference in its entirety. While notwishing to be bound by theory, in some embodiments, the membrane shieldsthe cells from the immune system and/or keeps a plurality of cells inproximity, facilitating interaction with each other or each other'sproducts.

Methods of Treatment with Compositions Herein, e.g., Enucleated RedBlood Cells

Methods of administering enucleated red blood cells (e.g.,reticulocytes) comprising (e.g., expressing) exogenous agent (e.g.,polypeptides) are described, e.g., in WO2015/073587 and WO2015/153102,each of which is incorporated by reference in its entirety.

In embodiments, the enucleated red blood cells described herein areadministered to a subject, e.g., a mammal, e.g., a human. Exemplarymammals that can be treated include without limitation, humans, domesticanimals (e.g., dogs, cats and the like), farm animals (e.g., cows,sheep, pigs, horses and the like) and laboratory animals (e.g., monkey,rats, mice, rabbits, guinea pigs and the like). The methods describedherein are applicable to both human therapy and veterinary applications.

In some embodiments, the RBCs are administered to a patient every 1, 2,3, 4, 5, or 6 months.

In some embodiments, a dose of RBC comprises about 1×10⁹-2×10⁹,2×10⁹-5×10⁹, 5×10⁹-1×10¹⁰, 1×10¹⁰-2×10¹⁰, 2×10¹⁰-5×10¹⁰, 5×10¹⁰-1×10¹¹,1×10¹¹-2×10¹¹, 2×10¹¹-5×10¹¹, 5×10¹¹-1×10¹², 1×10¹²-2×10¹²,2×10¹²-5×10¹², or 5×10¹²-1×10¹³ cells.

In some embodiments, the RBCs are administered to a patient in a dosingregimen (dose and periodicity of administration) sufficient to maintainfunction of the administered RBCs in the bloodstream of the patient overa period of 2 weeks to a year, e.g., one month to one year or longer,e.g., at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, ayear, 2 years.

In some aspects, the present disclosure provides a method of treating adisease or condition described herein, comprising administering to asubject in need thereof a composition described herein, e.g., anenucleated red blood cell (e.g., reticulocyte) described herein. In someembodiments, the disease or condition is a cancer. In some aspects, thedisclosure provides a use of an erythroid cell, e.g., red blood cell,described herein for treating a disease or condition described herein,e.g., a cancer. In some aspects, the disclosure provides a use of anerythroid cell, e.g., red blood cell described herein for manufacture ofa medicament for treating a disease or condition described herein, e.g.,a cancer.

Types of cancer include acute lymphoblastic leukaemia (ALL), acutemyeloid leukaemia (AML), anal cancer, bile duct cancer, bladder cancer,bone cancer, bowel cancer, brain tumours, breast cancer, cancer ofunknown primary, cancer spread to bone, cancer spread to brain, cancerspread to liver, cancer spread to lung, carcinoid, cervical cancer,choriocarcinoma, chronic lymphocytic leukaemia (CLL), chronic myeloidleukaemia (CML), colon cancer, colorectal cancer, endometrial cancer,eye cancer, gallbladder cancer, gastric cancer, gestationaltrophoblastic tumours (GTT), hairy cell leukaemia, head and neck cancer,Hodgkin lymphoma, kidney cancer, laryngeal cancer, leukaemia, livercancer, lung cancer, NSCLC, lymphoma, melanoma skin cancer,mesothelioma, men's cancer, molar pregnancy, mouth and oropharyngealcancer, myeloma, nasal and sinus cancers, nasopharyngeal cancer,non-Hodgkin lymphoma (NHL), oesophageal cancer, ovarian cancer,pancreatic cancer, penile cancer, prostate cancer, rare cancers, rectalcancer, salivary gland cancer, secondary cancers, skin cancer(non-melanoma), soft tissue sarcoma, stomach cancer, testicular cancer,thyroid cancer, unknown primary cancer, uterine cancer, vaginal cancer,and vulval cancer.

Additional Tables

TABLE 4 Exemplary modifiers, e.g., proteases Modifier Exemplary targetProteases IdeS IgG IdeZ (an immunoglobulin-degrading enzyme from IgGStreptococcus equi subspecies zooepidemicus) IgA protease IgG Papain IgGADAM17/TACE TNF-alpha mesotrypsin Peptides comprising linkages involvingthe carboxyl group of lysine or arginine Lysozyme peptidoglycanEndolysin peptidoglycan Endoproteinase, e.g., LysC (can cleave proteinson Protein having a Lys-Xaa motif C-terminal side of lysine residues)Metalloendopeptidase, e.g., LysN (can cleave Protein having an Xaa-Lysmotif proteins on amino side of lysine residues) Elastase, e.g.,Pseudomonas elastase (PaE) C3 alkaline protease (PaAP) C3 56 kDaprotease from Serratia marcescens C5a, C1-INH, alpha 2-antiplasmin,antithrombin III C5a peptidase, e.g., Streptocoocal C5a peptidase, C5aScpA, ScpB, SCPA Plasmin IgG, C3b, iC3b cysteine protease, e.g.,Streptococcal pyrogenic IgG, cytokines, extracellular matrix exotoxin B(SpeB) proteins PrtH (e.g., from Porphyromonas) IgG or C3 Staphylokinaseplasminogen, IgG, C3b Matrix metalloproteinases (e.g., MMP1, MMP2, ECMproteins, e.g., collagen, gelatin, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11,fibronectin, laminin, aggrecan, elastin, MMP12, MMP13, MMP14, MMP15,MMP16, fibrin MMP17, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25,MMP26, MMP27, MMP28) Other modifiers Protein disulfide isomerasesProteins comprising two cysteine residues Glycosyltransferases, e.g.,α-glucan-branching Protein comprising tyrosine, serine,glycosyltransferase, enzymatic branching factor, threonine, orasparagine glycosylation site branching glycosyltransferase, enzyme Q,glucosan transglycosylase, glycogen branching enzyme, amylose isomerase,plant branching enzyme, α-1,4- glucan: α-1,4-glucan-6-,glycosyltransferase, starch branching enzyme,UDP-N-acetyl-D-galactosamine, polypeptide,N-acetylgalactosaminyltransferase, GDP-fucose proteinO-fucosyltransferase 2, O- GlcNAc transferase Acetyltransferases ordeacetylases, e.g., histone nucleosome-histone acetyltransferase,histone acetokinase, histone acetylase, histone transacetylase, histonedeacetylase Acyltransferases Protein comprising an acyl groupPhosphatases, e.g., protein-tyrosine-phosphatase, phosphoproteinphosphotyrosine phosphatase, phosphoprotein phosphatase(phosphotyrosine), phosphotyrosine histone phosphatase, proteinphosphotyrosine phosphatase, tyrosylprotein phosphatase, phosphotyrosineprotein phosphatase, phosphotyrosylprotein phosphatase, tyrosine O-phosphate phosphatase, PPT-phosphatase, PTPase, [phosphotyrosine]proteinphosphatase, PTP- phosphatase Kinases, e.g., non-specificserine/threonine protein Protein comprising a serine or threoninekinase, Fas-activated serine/threonine kinase, phosphorylation siteGoodpasture antigen-binding protein kinase, IκB kinase, cAMP-dependentprotein kinase, cGMP- dependent protein kinase, protein kinase C, polokinase, cyclin-dependent kinase, mitogen-activated protein kinase,mitogen-activated protein kinase kinase kinase, receptor proteinserine/threonine kinase, dual-specificity kinase Gamma-carboxylasesProtein comprising glutamic acid Methyltransferases Protein comprising alysine methylation site; DNA; RNA Complement-factor inactivating moiety,e.g., Complement factor, e.g., C1, C2a, C4b, complement control protein,Factor H or Factor I C3, C3a, C3b, C5, C5a, C5b, C6, C7, C8, or C9

EXAMPLES Example 1. Agent-Synergistic Activity of eRBC Expressing TwoDifferent TRAIL Receptor Ligands on the Surface

The genes for TRAIL receptor agonists DR4.2 (SEQ ID 2) and DR5.2 (SEQ ID5) were synthesized. The genes were cloned into a lentivirus vector(SBI) upstream of the gene for human glycophorin A and separated by asequence encoding a 12-amino acid Gly-Ser (GGGSGGGSGGGS (SEQ ID NO: 19))flexible linker and an HA epitope tag (YPYDVPDY (SEQ ID NO: 20)).

Human CD34+ cells derived from mobilized peripheral blood cells fromnormal human donors were purchased frozen from AllCells Inc. Cells werethawed in PBS with 1% FBS. Cells were then cultured in StemSpan SFEMmedia with StemSpan CC100 Cytokine Mix at a density of 1E5 cells/mL.Media was swapped to differentiation media on day 5.

Virus production protocol was conducted as follows. Briefly, HEK293Tcells were seeded 24 hours before transfection. Cells were transfectedwith lentivector containing the construct along with packaging plasmids.A media swap was performed 24 hours after transfection and viruses wereharvested 72 hours after transfection. On day 6 after thaw, cells weretransduced with equal volumes of each virus in a 1:1 cell volume tovirus volume ratio, and spinoculated at 845×g for 1.5 hours with 5-10μg/ml of polybrene.

Transduced cells were differentiated in defined media to erythroidlineage cells and to mature enucleated reticulocytes following themethod of Hu et al., Blood 18 Apr. 2013 Vol 121, 16. In brief, the cellculture procedure was comprised of 3 phases. Composition of the baseculture medium was Iscove's Modified Dulbecco's Medium, 2% humanperipheral blood plasma, 3% human AB serum, 200 mg/mL Holohumantransferrin, 3 IU/mL heparin, and 10 mg/mL insulin. In the first phase(day 0 to day 6), CD341 cells at a concentration of 10{circumflex over( )}5/mL were cultured in the presence of 10 ng/mL stem cell factor, 1ng/mL IL-3, and 3 IU/mL erythropoietin. In the second phase (day 7 today 11), IL-3 was omitted from the culture medium. In the third phasethat lasted until day 21, the cell concentration was adjusted to10{circumflex over ( )}6/mL on day 11 and to 5×10{circumflex over( )}6/mL on day 15, respectively. The medium for this phase was the basemedium plus 3 IU/mL erythropoietin, and the concentration of transferrinwas adjusted to 1 mg/mL.

Expression of the transgenes was monitored by labeling with solubleTRAIL R1 and TRAIL R2 (purchased from Sigma-Aldrich Inc.) that had beenchemically conjugated to complementary fluorescent dyes Fluorescein andDyLight 650 and staining by flow cytometry. Expression levels of bothligands DR4.2 and DR5.2 were verified through flow cytometry.

A tumor cell line apoptosis assay was conducted according to a modifiedversion of Marconi et al., Cell Death and Disease 2013. In short, fullymature enucleated reticulocytes expressing DR4.2 and DR5.2 wereincubated with CFSE-labeled Raji Cells for 24 hours at a 1:1 ratio.Afterwards cells were stained with annexin V and analyzed by flowcytometry. Apoptosis percentages were determined from CFSE positive Rajicells that also stained positive for annexin V.

As shown in FIG. 1, when CFSE-labeled Raji cells were incubated withuntransduced, DR4.2 transduced, DR5.2 transduced, or a mixture of DR4.2transduced and DR5.2 transduced cultured reticulocytes, minimal celldeath was observed over background. However, when CFSE-labeled Rajicells were incubated with cultured reticulocytes that had beensimultaneously transduced with both DR4.2 and DR5.2 and thus expressboth proteins simultaneously, a significant amount of cell death wasobserved (equivalent to the maximal amount of TRAIL-induced apoptosisachievable in this assay with Raji cells—see, e.g. Marconi et al., CellDeath and Disease 2013). This data indicates that the coordinated actionof TRAIL receptor agonists on the surface of a single engineered redblood cell is able to induce cell killing in a synergistic manner,relative to cells expressing single TRAIL receptor agonists and even amixture of cells that each express a different TRAIL receptor agonist.

The cell population may be formulated in AS-3 additive solution andadministered intravenously to a patient suffering from Burkitt'sLymphoma. It is anticipated that the patient then exhibits animprovement in his symptoms as measured by reduction in lymph node size,improvement in hepatosplenomegaly, and/or reduction of nausea andvomiting.

Example 2. Generation of Capture eRBC Comprising 5 Cytokines for Use inTreating Sepsis

The genes for anti-TNFa (SEQ ID 7), anti-IL6 (SEQ ID 6), CD14 (Uniprot #P08571), IFNGR1 (Uniprot # P15260), and IL12R1 (Uniprot # P42701) aresynthesized by a commercial vendor. The genes are cloned into alentivirus vector (SBI) upstream of the gene for human glycophorin A andseparated by a sequence encoding a 12-amino acid Gly-Ser (GGGSGGGSGGGS(SEQ ID NO: 19)) flexible linker and an HA epitope tag (YPYDVPDY (SEQ IDNO: 20)).

Human CD34+ cells can be cultured, and virus can be produced, asdescribed in Example 1. Transduced cells are differentiated as describedherein.

To assess the expression of the transgenes, cells are labeledsimultaneously with the ligands TNFa, IL-6, IFNg, and IL-12 (purchasedfrom Life Technologies), as well as lipopolysaccharide (ThermoFisher),that are chemically conjugated to complementary fluorescent dyes. Thecells are analyzed by flow cytometry to verify that (a) the agents areall expressed on the surface of the cell and (b) the agents are capableof binding to their target ligands.

The cell population is formulated in AS-3 additive solution andadministered intravenously to a patient who is developing sepsis. It isanticipated that the patient then exhibits an improvement in hissymptoms as measured by a reduction in circulating cytokine levels, areduction or prevention of vascular leak syndrome, and improvedsurvival.

Example 3. eRBC Comprising Combinatorial Library of Tumor Antigens forUse as Cancer Vaccine

Human CD34+ cells can be cultured as described in Example 1. Cells aredifferentiated to erythroid lineage cells as described herein.

A sample of melanoma cancer cells is isolated from a patient by biopsy.The cells are lysed and total RNA is extracted using a silica columnpurification (ThermoFisher), quantified for RNA content by absorbancespectroscopy, and stored at −80 C.

Four days before terminal differentiation of the red blood cell culture,cells are collected, washed twice with serum-free IMDM, and resuspendedto a final concentration of 10-40×10{circumflex over ( )}6 cells/mL inOpti-MEM. Subsequently, 0.5 mL of the cell suspension is mixed with 20ug of mRNA, and electroporated in a 0.4-cm cuvette using an EasyjectPlus device (EquiBio, Kent, United Kingdom) at conditions of 300V and150 uF. After electroporation, fresh red blood cell maturation medium isadded to the cell suspension and cells are further incubated at 37° C.in a humidified atmosphere supplemented with 5% CO2.

Fully mature reticulocytes are characterized for protein expression bymass spectrometric analysis of cell lysate. Non-electroporated cells andelectroporated cells that are administered PBS instead of RNA are usedas controls to identify endogenous reticulocyte proteins from exogenouscancer-derived proteins.

The cell population is formulated in AS-3 additive solution andadministered intravenously to a patient who suffering from melanoma. Itis anticipated that the patient then exhibits an immune response againstthe melanoma antigens, measured by reduction in metastatic masses by CTscanning and resolution of melanoma skin lesions.

Example 4: Genetic Engineering of Erythrocytes as an Anti-Tumor Therapyfor Non-Hodgkin's Lymphoma (NHL)

Red blood cells were generated that express on their surface antibodiesagainst PD-1 and PD-L1 (RCT-antiPD-1 and RCT-antiPD-L1) to assesswhether these cells could bind their respective targets and activate arobust immune response. Binding of RCT-antiPD-1 and RCT-antiPD-L1 torecombinant PD-1 and PD-L1, respectively was determined using flowcytometry, and was shown to be highly specific. Red blood cells werealso produced which express on their surface a fusion proteincomprising, from N-to-C terminus, an ipilimumab-based anti-CTLA4 scFvantibody domain, an epitope tag, and full-length GPA (extracellular,transmembrane, and cytoplasmic domains. Robust expression of anti-CTLA4polypeptides was observed in a flow cytometry assay, with over 95.2% ofcells expressed anti-CTLA4 after transfection with a vector encodinganti-CTLA4.

Functional activity was tested using an in vitro Jurkat cell IL-2secretion assay. In this assay, IL-2 secretion is inhibited byincubating Jurkat cells with NHL cells (Z138) expressing PD-L1 inducedby stimulation with CD3/CD28 tetramers. IL-2 secretion was rescued byculturing the Jurkat and Z138 cells with RCT-antiPD-1 or RCT-antiPD-L1but not control RCT. RCT-antiCTLA4 also showed a rescue and restorationof T cell IL-2 secretion.

The ability of these engineered red blood cells to elicit activation ina standard antigen recall assay was assayed. A robust 4-6 fold increasewas demonstrated in interferon-gamma secretion of peripheral bloodmononuclear cells (PBMC) in an antigen recall assay. Donor PBMC werestimulated with a common flu virus antigen. Memory T cells sensitive toimmune checkpoint inhibition were tested for activation and gammainterferon secretion by co-culture with RCT-antiPD-1 or RCT-antiPD-L1 incomparison to control PBMCs or control RCT.

These experiments indicate that red blood cells are capable of engagingin specific cell-cell interactions and engaging the immune checkpoint.

In addition, red blood cells expressing an anti-CD20 single chainvariable fragment on their surface (RCT-antiCD20) were generated. Theirability to bind CD20+ lymphoma cells in vitro was assessed. Thisexperiment demonstrated efficient and specific binding of RCT-antiCD20to target cells using flow cytometry and immunofluorescent microscopy.It was also assessed whether this interaction could induce apoptosis, byco-culturing RCT-antiCD20 cells with a panel of CD20+ human lymphomacell lines, representing lymphoma subtypes; DoHH2 (follicular lymphoma),Ramos (Burkitt's lymphoma), Granta-519 (Mantle Cell Lymphoma) andSU-DHL-4 (diffuse large B cell lymphoma). In all cases, RCT-antiCD20co-culture resulted in increased apoptosis relative to RCT or solubleRituximab monoclonal antibody alone. Direct tumor cell killing in vitrois hypothesized to be more effective than monoclonal antibody alone dueto the hyper-crosslinking of CD20 on the lymphoma cell. This effect wasshown both by in situ demonstration of receptor clustering and by astimulation of apoptotic pathways. These findings therefore demonstratea novel biology for proteins expressed on RCT and warranted testing forimpact on lymphoma tumors in vivo.

Example 5: Capture and Modification of a Target Protein

In this Example, transgenic enucleated erythroid cells were used tocapture and modify a target protein. The control cells and theexperimental cells each comprise endogenous glycophorin A (GPA) in theirmembranes, which was used to bind the target protein. The experimentalcells expressed an exogenous protein comprising surface-exposed IdeSfused to GPA as a membrane anchor. IdeS is capable of cleavingantibodies to produce a F(ab′)2 fragment and a Fc fragment. The targetprotein is an anti-GPA antibody that is fluorescently labelled withFITC. Both the constant and variable regions of the target antibody wereFITC-labelled, so that if the antibody was cleaved, both fragments couldbe detected.

First, the control cells and IdeS-expressing cells were tested by FACSfor the ability to bind the anti-GPA antibody. Both control andIdeS-expressing cells bound the antibody as measured by association ofFITC with the cells (data not shown). In addition, both control andIdeS-expressing cells bound the antibody as measured by or using asecond detection method with a fluorescently labeled anti-rabbit Fcantibody (data not shown). These measurements were taken at an earlytimepoint, before cells were incubated to allow IdeS-mediated cleavageof the target antibody.

In contrast, only the IdeS-expressing cells were able to cleave thetarget antibody. This was shown by incubating the control orIdeS-expressing cells with the target antibody to allow antibodycleavage to occur. Fluorescently labeled anti-rabbit Fc antibody wasadded to the reaction in order to detect intact antibodies on thesurface of the erythroid cells. The IdeS-expressing cells showed adecrease in anti-rabbit Fc association with the cells (FIG. 2),indicating lower levels of Fc on the surface of the IdeS-expressingcells compared to the control cells. There was no decrease in the amountof the directly FITC-labeled target antibody associated with controlcells or IdeS-expressing cells, indicating that at least theFITC-labeled variable region of the target antibody still bound theIdeS-expressing and control cells. This result was confirmed by Westernblot, where anti rabbit heavy chain and anti rabbit light chainantibodies were used to detect intact and cleaved antibody in thesupernatant of control or IdeS-expressing cells. The experiment showedthat IdeS-expressing erythroid cells but not control erythroid cellscleaved the anti-GPA-antibody, resulting in appearance of the heavychain fragment (FIG. 3).

Thus, the control cells were able to bind the target antibody, but onlythe IdeS-expressing cells were able to bind and cleave the targetantibody.

Example 6: RCT-Anti-PD-L1 Promotes T Cell Proliferation

This Example demonstrates that co-culture of RCT-antiPD-L1 with PBMC hasled to enhanced T-cell proliferation, based upon a 4.4 fold increase intotal count of T cells following incubation with RCT-antiPD-L1 whencompared to PBMCs alone.

Example 7: RCTs Expressing a Costimulatory Protein

Approaching T-cell activation from another angle, RCTs were engineeredto express 41-BB-L, a co-stimulatory protein that is expressed onantigen presenting cells and binds the 41-BB receptor on T-cells(RCT-41-BB-L). Binding of RCT-41-BB-L to recombinant 41-BB wasdetermined using flow cytometry. Co-culture of PBMCs with RCT-41-BB-Lshowed a 1.7 fold increase in T-cell proliferation compared to PBMCsalone. Finally, when RCT-41-BB-L were incubated with Jurkat cellsoverexpressing 41-BB and NFkB-Luc2P, activation of NFkB-mediatedluciferase expression increased 30 fold compared to controls.

Example 8: Red Cell Therapeutics Co-Expressing Anti-CD20 and TRAILLigand

When erythroid cells were engineered to simultaneously express anti-CD20as well as Trail ligand (an apoptosis inducing agent), co-culture ofRamos cells with RCT-antiCD20, RCT-Trail, and RCT-antiCD20+ Trail(co-expressed) exert 32%, 47% and 76% apoptosis respectively after 48hours, suggesting a synergistic cell-killing effect of the co-expressingRCTs.

A cell population comprising TRAIL ligand and anti-CD20 moiety may beformulated in AS-3 additive solution and administered intravenously to apatient, e.g., a patient suffering from a cancer.

The invention claimed is:
 1. A genetically engineered enucleatederythroid cell comprising an exogenous polypeptide comprising a4-1BB-binding fragment of 4-1BBL at the surface of the geneticallyengineered enucleated erythroid cell, wherein the enucleated erythroidcell was produced by a process comprising: introducing an exogenousnucleic acid encoding the exogenous polypeptide into a nucleatederythroid cell, or a precursor thereof; and culturing the nucleatederythroid cell under conditions suitable for enucleation and forproduction of the exogenous polypeptide.
 2. The genetically engineeredenucleated erythroid cell of claim 1, wherein the exogenous polypeptidefurther comprises a transmembrane domain.
 3. The genetically engineeredenucleated erythroid cell of claim 2, wherein the transmembrane domaincomprises a polypeptide having at least 80% identity to the amino acidsequence of SEQ ID NO:
 9. 4. The genetically engineered enucleated cellof claim 1, wherein the exogenous polypeptide comprises a 4-1BB-bindingfragment of an amino acid sequence of SEQ ID NO:
 21. 5. The geneticallyengineered enucleated erythroid cell of claim 1, wherein the exogenouspolypeptide lacks a sortase transfer signature.
 6. The geneticallyengineered enucleated erythroid cell of claim 1, which comprises aplurality of exogenous fusion proteins on the surface of the geneticallyengineered enucleated erythroid cell, wherein at least 50% of theexogenous fusion proteins comprised by the cell have an identicalsequence.
 7. The genetically engineered enucleated erythroid cell ofclaim 1, which is a reticulocyte.
 8. A method of treating cancer,comprising administering the genetically engineered enucleated erythroidcell of claim 1 to a subject in need thereof.
 9. The method of claim 8,wherein the cancer is selected from lung cancer, NSCLC, esophagealcancer, bladder cancer, stomach cancer, head and neck cancer, colorectalcancer, uterine cancer, cervical cancer, breast cancer, prostate cancer,ovarian cancer, liver cancer, kidney cancer, myeloma, and non-Hodgkinlymphoma.
 10. The genetically engineered enucleated erythroid cell ofclaim 2, wherein the transmembrane domain comprises a type I red bloodcell transmembrane domain.
 11. The genetically engineered enucleatederythroid cell of claim 2, wherein the transmembrane domain comprises atype II red blood cell transmembrane domain.
 12. The geneticallyengineered enucleated erythroid cell of claim 1, which is capable ofpromoting T cell proliferation.
 13. The genetically engineeredenucleated erythroid cell of claim 1, wherein the transmembrane domaincomprises a glycophorin A (GPA) transmembrane domain.
 14. Thegenetically engineered enucleated erythroid cell of claim 1, whichfurther comprises a second exogenous protein.
 15. The geneticallyengineered enucleated erythroid cell of claim 1, wherein the culturingcomprises expanding the nucleated erythroid cell at least 1,000-fold.16. A population of cells comprising a plurality of geneticallyengineered enucleated erythroid cells of claim 1, wherein at least 60%of cells in the population are enucleated erythroid cells.
 17. Thepopulation of claim 16, wherein at least 90% of cells in the populationare enucleated erythroid cells.
 18. The population of claim 16, whereinat least 80% of cells in the population are enucleated erythroid cellsthat comprise the exogenous polypeptide.
 19. A pharmaceuticalpreparation comprising a plurality of genetically engineered enucleatederythroid cells of claim
 1. 20. The genetically engineered enucleatederythroid cell of claim 1, wherein the precursor is a CD34+ cell.